Endre søk
Begrens søket
1234567 151 - 200 of 502
RefereraExporteraLink til resultatlisten
Permanent link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Treff pr side
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sortering
  • Standard (Relevans)
  • Forfatter A-Ø
  • Forfatter Ø-A
  • Tittel A-Ø
  • Tittel Ø-A
  • Type publikasjon A-Ø
  • Type publikasjon Ø-A
  • Eldste først
  • Nyeste først
  • Skapad (Eldste først)
  • Skapad (Nyeste først)
  • Senast uppdaterad (Eldste først)
  • Senast uppdaterad (Nyeste først)
  • Disputationsdatum (tidligste først)
  • Disputationsdatum (siste først)
  • Standard (Relevans)
  • Forfatter A-Ø
  • Forfatter Ø-A
  • Tittel A-Ø
  • Tittel Ø-A
  • Type publikasjon A-Ø
  • Type publikasjon Ø-A
  • Eldste først
  • Nyeste først
  • Skapad (Eldste først)
  • Skapad (Nyeste først)
  • Senast uppdaterad (Eldste først)
  • Senast uppdaterad (Nyeste først)
  • Disputationsdatum (tidligste først)
  • Disputationsdatum (siste først)
Merk
Maxantalet träffar du kan exportera från sökgränssnittet är 250. Vid större uttag använd dig av utsökningar.
  • 151. Graslund, S.
    et al.
    Larsson, M.
    Falk, R.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoog, C.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Single-vector three-frame expression systems for affinity-tagged proteins2002Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 215, nr 1, s. 139-147Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.

  • 152. Gremel, Gabriela
    et al.
    Bergman, Julia
    Djureinovic, Dijana
    Edqvist, Per-Henrik
    Maindad, Vikas
    Bharambe, Bhavana M.
    Khan, Wasif Ali Z. A.
    Navani, Sanjay
    Elebro, Jacob
    Jirström, Karin
    Hellberg, Dan
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Micke, Patrick
    Pontén, Fredrik
    A systematic analysis of commonly used antibodies in cancer diagnostics2014Inngår i: Histopathology, ISSN 0309-0167, E-ISSN 1365-2559, Vol. 64, nr 2, s. 293-305Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    AimsImmunohistochemistry plays a pivotal role in cancer differential diagnostics. To identify the primary tumour from a metastasis specimen remains a significant challenge, despite the availability of an increasing number of antibodies. The aim of the present study was to provide evidence-based data on the diagnostic power of antibodies used frequently for clinical differential diagnostics. Methods and resultsA tissue microarray cohort comprising 940 tumour samples, of which 502 were metastatic lesions, representing tumours from 18 different organs and four non-localized cancer types, was analysed using immunohistochemistry with 27 well-established antibodies used in clinical differential diagnostics. Few antibodies, e.g. prostate-specific antigen and thyroglobulin, showed a cancer type-related sensitivity and specificity of more than 95%. A majority of the antibodies showed a low degree of sensitivity and specificity for defined cancer types. Combinations of antibodies provided limited added value for differential diagnostics of cancer types. ConclusionsThe results from analysing 27 diagnostic antibodies on consecutive sections of 940 defined tumours provide a unique repository of data that can empower a more optimal use of clinical immunohistochemistry. Our results highlight the benefit of immunohistochemistry and the unmet need for novel markers to improve differential diagnostics of cancer.

  • 153. Gremel, Gabriela
    et al.
    Djureinovic, Dijana
    Niinivirta, Marjut
    Laird, Alexander
    Ljungqvist, Oscar
    Johannesson, Henrik
    Bergman, Julia
    Edqvist, Per-Henrik
    Navani, Sanjay
    Khan, Naila
    Patil, Tushar
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Harrison, David J.
    Ullenhag, Gustav J.
    Stewart, Grant D.
    Ponten, Fredrik
    A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma2017Inngår i: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 17, artikkel-id 9Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). Methods: Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray (n = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts (n = 181; n = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. Results: CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203-0.719, P = 0.003). Conclusions: CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.

  • 154. Gremel, Gabriela
    et al.
    Wanders, Alkwin
    Cedernaes, Jonathan
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Edlund, Karolina
    Sjostedt, Evelina
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    The human gastrointestinal tract-specific transcriptome and proteome as defined by RNA sequencing and antibody-based profiling2015Inngår i: Journal of gastroenterology, ISSN 0944-1174, E-ISSN 1435-5922, Vol. 50, nr 1, s. 46-57Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The gastrointestinal tract (GIT) is subdivided into different anatomical organs with many shared functions and characteristics, but also distinct differences. We have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to describe the gene and protein expression patterns that define the human GIT. RNA sequencing data derived from stomach, duodenum, jejunum/ileum and colon specimens were compared to gene expression levels in 23 other normal human tissues analysed with the same method. Protein profiling based on immunohistochemistry and tissue microarrays was used to sub-localize the corresponding proteins with GIT-specific expression into sub-cellular compartments and cell types. Approximately 75 % of all human protein-coding genes were expressed in at least one of the GIT tissues. Only 51 genes showed enriched expression in either one of the GIT tissues and an additional 83 genes were enriched in two or more GIT tissues. The list of GIT-enriched genes with validated protein expression patterns included various well-known but also previously uncharacterised or poorly studied genes. For instance, the colon-enriched expression of NXPE family member 1 (NXPE1) was established, while NLR family, pyrin domain-containing 6 (NLRP6) expression was primarily found in the human small intestine. We have applied a genome-wide analysis based on transcriptomics and antibody-based protein profiling to identify genes that are expressed in a specific manner within the human GIT. These genes and proteins constitute important starting points for an improved understanding of the normal function and the different states of disease associated with the GIT.

  • 155.
    Gry, Marcus
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). Uppsala University, Sweden.
    Oksvold, Per
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Pontén, F.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Tissue-specific protein expression in human cells, tissues and organs2010Inngår i: Journal of Proteomics & Bioinformatics, ISSN 0974-276X, E-ISSN 0974-276X, Vol. 3, nr 10, s. 286-293Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An important part of understanding human biology is the study of tissue-specific expression both at the gene and protein level. In this study, the analysis of tissue specific protein expression was performed based on tissue micro array data available on the public Human Protein Atlas database (www.proteinatlas.org). An analysis of human proteins, corresponding to approximately one third of the protein-encoding genes, was carried out in 65 human tissues and cell types. The spatial distribution and relative abundance of 6,678 human proteins, were analyzed in different cell populations from various organs and tissues in the human body using unsupervised methods, such as hierarchical clustering and principal component analysis, as well as with supervised methods (Breiman, 2001). Well-known markers, such as neuromodulin for the central nervous system, keratin 20 for gastrointestinal tract and CD45 for hematopoietic cells, were identified as tissue-specific. Proteins expressed in a tissue-specific manner were identified for cells in all of the investigated tissues, including the central nervous system, hematopoietic system, squamous epithelium, mesenchymal cells and cells from the gastrointestinal tract. Several proteins not yet associated with tissue-specificity were identified, providing starting points for further studies to explore tissue-specific functions. This includes proteins with no known function, such as ZNF509 expressed in CNS and C1orf201 expressed in the gastro-intestinal tract. In general, the majority of the gene products are expressed in a ubiquitous manner and few proteins are detected exclusively in cells from a particular tissue class, as exemplified by less than 1% of the analyzed proteins found only in the brain.

  • 156.
    Gry, Marcus
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Rimini, Rebecca
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Strömberg, Sara
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital.
    Asplund, Anna
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital.
    Pontén, Fredrik
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Correlations between RNA and protein expression profiles in 23 human cell lines2009Inngår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 10Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays) and protein profiles (determined immunohistochemically in tissue microarrays) of 1066 gene products in 23 human cell lines. Results: A high mean correlation coefficient (0.52) was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion: Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies’ specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  • 157.
    Gräslund, Susanne
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Eklund, Malin
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Falk, Ronny
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Nygen, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Ståhl, Stefan
    KTH, Tidigare Institutioner                               , Bioteknologi.
    A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, nr 1, s. 41-50Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

  • 158.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Ehn, Maria
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Gunnel, Lundin
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002Inngår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, nr 1-2, s. 157-166Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained α-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI≈5.8) and ZZ-Cutinase-Zbasic (pI≈7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

  • 159.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Hedhammar, My
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, nr 1, s. 93-102Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

  • 160.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Lundin, Gunnel
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Charge engineering of a protein domain to allow efficient ion-exchange recovery2000Inngår i: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, nr 10, s. 703-709Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

  • 161.
    Gräslund, Torbjörn
    et al.
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Nilsson, Joakim
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Lindberg, Michael
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein1997Inngår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, s. 125-132Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I fromThermus aquaticus.The DNA polymerase ([Delta]Taq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the [Delta]TaqDNA polymerase, affinity-purified ABP-[Delta]Taqcould be heat-eluted from HSA columns by incubation at 85ï¿œC. To produce free [Delta]TaqDNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure [Delta]TaqDNA polymerase with full enzymatic activity.

  • 162. Gulich, S.
    et al.
    Linhult, M.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Stability towards alkaline conditions can be engineered into a protein ligand2000Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 80, nr 2, s. 169-178Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.

  • 163. Gulich, S.
    et al.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography2000Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, nr 03-feb, s. 233-244Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.

  • 164. Gustafsson, A. C.
    et al.
    Kijas, J. M. H.
    Alderborn, A.
    Uhlén, Mathias
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Andersson, L.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing2001Inngår i: Animal Biotechnology, ISSN 1049-5398, E-ISSN 1532-2378, Vol. 12, nr 2, s. 145-153Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The increasing interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) emphasis the need for high-throughput and cost effective scoring methods. Pyrosequencing is a novel method for screening SNPs. In this study we examine breed specific SNPs in the pig melanocortin I receptor gene (MC1R), some causing coat color phenotypes. A total of fifteen pigs representing eight breeds and crosses were analyzed by pyrosequencing. In addition to nine previously known SNPs, we also detected one new missense mutation by pyrosequencing. We here show that the SNPs were readily scored using standard reaction conditions. Insertions as well as substitutions were unambiguously detected and all genotypes were resolved in terms of homo- and heterozygozity.

  • 165. Gustavsson, Elin
    et al.
    Ek, Sara
    Steen, Johanna
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Kristensson, Malin
    Älgenäs, Cajsa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Wingren, Christer
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Borrebaeck, Carl A. K.
    Surrogate antigens as targets for proteome-wide binder selection2011Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 28, nr 4, s. 302-311Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

  • 166. Haab, B. B.
    et al.
    Paulovich, A. G.
    Leigh Anderson, N.
    Clark, A. M.
    Downing, G. J.
    Hermajakob, H.
    LaBaer, J.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    A reagent resource to identify proteins and peptides of interest for the cancer community2006Inngår i: Molecular and Cellular Proteomics, ISSN 1535-9476, Vol. 5, nr 10, s. 1996-2007Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    On the basis of discussions with representatives from all sectors of the cancer research community, the National Cancer Institute (NCI) recognizes the immense opportunities to apply proteomics technologies to further cancer research. Validated and well characterized affinity capture reagents (e.g. antibodies, aptamers, and affibodies) will play a key role in proteomics research platforms for the prevention, early detection, treatment, and monitoring of cancer. To discuss ways to develop new resources and optimize current opportunities in this area, the NCI convened the "Proteomic Technologies Reagents Resource Workshop" in Chicago, IL on December 12-13, 2005. The workshop brought together leading scientists in proteomics research to discuss model systems for evaluating and delivering resources for reagents to support MS and affinity capture platforms. Speakers discussed issues and identified action items related to an overall vision for and proposed models for a shared proteomics reagents resource, applications of affinity capture methods in cancer research, quality control and validation of affinity capture reagents, considerations for target selection, and construction of a reagents database. The meeting also featured presentations and discussion from leading private sector investigators on state-of-the-art technologies and capabilities to meet the user community's needs. This workshop was developed as a component of the NCI's Clinical Proteomics Technologies Initiative for Cancer, a coordinated initiative that includes the establishment of reagent resources for the scientific community. This workshop report explores various approaches to develop a framework that will most effectively fulfill the needs of the NCI and the cancer research community.

  • 167. Habuka, Masato
    et al.
    Fagerberg, Linn
    Hallstrom, Bjorn M.
    Ponten, Fredrik
    Yamamoto, Tadashi
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    The Urinary Bladder Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling2015Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 12, artikkel-id e0145301Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To understand functions and diseases of urinary bladder, it is important to define its molecular constituents and their roles in urinary bladder biology. Here, we performed genome-wide deep RNA sequencing analysis of human urinary bladder samples and identified genes upregulated in the urinary bladder by comparing the transcriptome data to those of all other major human tissue types. 90 protein-coding genes were elevated in the urinary bladder, either with enhanced expression uniquely in the urinary bladder or elevated expression together with at least one other tissue (group enriched). We further examined the localization of these proteins by immunohistochemistry and tissue microarrays and 20 of these 90 proteins were localized to the whole urothelium with a majority not yet described in the context of the urinary bladder. Four additional proteins were found specifically in the umbrella cells (Uroplakin 1a, 2, 3a, and 3b), and three in the intermediate/basal cells (KRT17, PCP4L1 and ATP1A4). 61 of the 90 elevated genes have not been previously described in the context of urinary bladder and the corresponding proteins are interesting targets for more in-depth studies. In summary, an integrated omics approach using transcriptomics and antibody-based profiling has been used to define a comprehensive list of proteins elevated in the urinary bladder.

  • 168.
    Habuka, Masato
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska University Hospital, Sweden .
    Fagerberg, Linn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kampf, Caroline
    Edlund, Karolina
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Yamamoto, Tadashi
    Pontén, Fredrik
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Odeberg, Jacob
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska University Hospital, Sweden.
    The Kidney Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling2014Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 12, s. e116125-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into tissue enriched, group enriched or tissue enhanced. Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n=11), proximal tubules (n=120), distal tubules (n=9) or collecting ducts (n=8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease.

  • 169. Hamsten, C.
    et al.
    Skattum, L.
    Truedsson, L.
    von Döbeln, U.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hammarström, L.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Heat differentiated complement factor profiling2015Inngår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 126, s. 155-162Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

  • 170.
    Hamsten, Carl
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Westberg, Joakim
    Bölske, Göran
    Ayling, Roger
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Persson, Anja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.2008Inngår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, s. 539-549Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

  • 171. Hedberg, Jesper
    et al.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Donnes, Pierre
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Molecular profiling of human kidney injury using antibody suspension bead arrays2009Inngår i: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 189, s. S94-S94Artikkel i tidsskrift (Annet vitenskapelig)
  • 172.
    Hedhammar, My
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Gräslund, Torbjörn
    KTH, Tidigare Institutioner, Bioteknologi.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Bioteknologi.
    Hober, Sophia
    KTH, Tidigare Institutioner, Bioteknologi.
    Negatively charged purification tags for selective anion-exchange recovery2004Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, nr 11, s. 779-786Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

     A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

  • 173.
    Hedhammar, My
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Stenvall, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Lönneborg, Rosa
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nord, Olof
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Sjölin, Olle
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Brismar, Hjalmar
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.
    Uhlén, Matthias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ottosson, Jenny
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    A Novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein2005Inngår i: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 119, nr 2, s. 133-146Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

  • 174. Hedner, Charlotta
    et al.
    Gaber, Alexander
    Korkocic, Dejan
    Nodin, Bjorn
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kuteeva, Eugenia
    Johannesson, Henrik
    Jirstrom, Karin
    Eberhard, Jakob
    SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma2014Inngår i: Virchows Archiv, ISSN 0945-6317, E-ISSN 1432-2307, Vol. 465, nr 6, s. 649-659Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype.

  • 175. Hertzberg, M.
    et al.
    Aspeborg, H.
    Schrader, J.
    Andersson, A.
    Erlandsson, R.
    Blomqvist, K.
    Bhalerao, R.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Bioteknologi.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner, Bioteknologi.
    Lundeberg, Joakim
    KTH, Tidigare Institutioner, Bioteknologi.
    Sundberg, B.
    Nilsson, Peter
    KTH, Tidigare Institutioner, Bioteknologi.
    Sandberg, G.
    A transcriptional roadmap to wood formation2001Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 98, nr 25, s. 14732-14737Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

  • 176. Hjalmarsson, S
    et al.
    Alderborn, A
    Fock, C
    Muldin, I
    Kling, H
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Bioteknologi.
    Engstrand, L
    Rapid combined characterization of microorganism and host genotypes using a single technology2004Inngår i: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 9, nr 2, s. 138-145Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background. Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrose-quencing(TM) technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. Materials and Methods. DNA from 87 clinical isolates of H. pylori 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. Results. All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. Conclusions. We conclude that genetic analysis using Pyrosequencing(TM) technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markets.

  • 177.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Brennan, Donal J
    Zendehrokh, Nooreldin
    Eberhard, Jakob
    Nodin, Björn
    Gaber, Alexander
    Pontén, Fredrik
    Johannesson, Henrik
    Smaragdi, Kristina
    Frantz, Christian
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Johnson, Louis B
    Påhlman, Sven
    Jirström, Karin
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer2011Inngår i: Proteomics. Clinical applications, ISSN 1862-8354, Vol. 5, nr 11-12, s. 624-35Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. Experimental design: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). Results: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. Conclusion and clinical relevance: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.

  • 178.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Inngår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 179.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Igel, Ulrika
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, Henrik
    Stadler, Charlotte
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Sjoberg, Anna
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Christine
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Generation of monospecific antibodies based on affinity capture of polyclonal antibodies2011Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 11, s. 1824-1835Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

  • 180.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopesManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 181.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 182.
    Hober, Andreas
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Edfors, Fredrik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Ryaboshapkina, Maria
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Malmqvist, Jonas
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Rosengren, Louise
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Percy, Andrew J.
    Cambridge Isotope Labs Inc, Dept Applicat Dev, Tewksbury, MA 01876 USA..
    Lind, Lars
    Uppsala Univ, Dept Med Sci, Uppsala, Sweden..
    Forsström, Björn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Oscarsson, Jan
    AstraZeneca, Global Med Dev Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Miliotis, Tasso
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden..
    Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay2019Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, nr 12, s. 2433-2446Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

  • 183.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hansson, A
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nilsson, B
    Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.1994Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 33, nr 22Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.

  • 184.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Lundström Ljung, J
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nilsson, B
    Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions.1999Inngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 443, nr 3Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance.

  • 185.
    Hober, Sophia
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Human protein atlas and the use of microarray technologies2008Inngår i: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 19, nr 1, s. 30-35Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies.

  • 186. Holgersson, G.
    et al.
    Ekman, S.
    Reizenstein, J.
    Bergqvist, M.
    Pontén, F.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Magnusson, K.
    Jonnalagadda, P.
    Asplund, A.
    Strömberg, S.
    Linder, A.
    Blomquist, E.
    Liljeholm, M.
    Löden, B.
    Hellström, K.
    Bergström, S.
    Molecular profiling using tissue microarrays as a tool to identify predictive biomarkers in laryngeal cancer treated with radiotherapy2010Inngår i: Cancer Genomics & Proteomics, ISSN 1109-6535, E-ISSN 1790-6245, Vol. 7, nr 1, s. 1-7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aim: To explore the usefulness of the expression of five potential cancer biomarkers in predicting outcome in patients with laryngeal cancer. Materials and Methods: In the present study, the Swedish National Cancer Registry databases were used to identify patients with laryngeal cancer diagnosed during the years 1978-2004 in the Uppsala-Örebro region and treated with radiotherapy. The expression of Ki-67, MutS homolog 2, (MSH2), p53, B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1 in the cancer cells was assessed immunohistochemically using tissue microarrays (TMAs) and its predictve value on survival and relapse was analyzed using Cox regression models. Results: A total of 39 patients were included in the present study. Nuclear MSH2 staining was statistically significantly correlated to Ki-67 expression (p=0.022). However, univariate and multivariate Cox analyses showed no statistically significant association between the expression of the investigated biomarkers and overall survival or relapse. Conclusion: The present exploratory study does not show any significant predictive value of the biomarkers examined with respect to survival or relapse. However, with larger patient cohorts, we believe that protein profiling using TMAs and immunohistochemistry is a feasible strategy for prognostic and predictive biomarker screening in laryngeal cancer.

  • 187. Holmberg, A.
    et al.
    Blomstergren, A.
    Nord, O.
    Lukacs, M.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures2005Inngår i: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, nr 3, s. 501-510Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K-d, in the order of 4 x 10(-14) m. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70degreesC can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluiclics and nanotechnology.

  • 188. Hu, Francis Jingxin
    et al.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restricted Ablation In vitro) for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improve- ments in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of intact antibody scFv genes and simultaneous diversification of all six CDRs. Here, we de- scribe the generation of mutagenesis libraries for antibody affinity maturation, using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. This procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, and elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with di- versity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed >99% functional diversity in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed quicker enrichment of improved binders compared to the other two diversification strategies.

  • 189.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Lundqvist, Magnus
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    Uhlén, Mathias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap. KTH, Centra, Science for Life Laboratory, SciLifeLab. Tech Univ Denmark, Novo Nord Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Rockberg, Johan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap, Proteinteknologi.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping2019Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, nr 6, artikkel-id e34Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • 190.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Lundqvist, Magnus
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    SPUX - A Solid Phase Uracil Excision Method for Antibody Affinity Maturation and Paratope MappingManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.

  • 191.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody2014Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, nr 1, s. 35-43Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

  • 192.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Persson, Helena
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Lund University, Sweden.
    Säll, Anna
    Borrebaeck, Carl
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders2017Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

  • 193.
    Hu, Francis Jingxin
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Volk, Anna-Luisa
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Persson, Helena
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Säll, Anna
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Borrebaeck, Carl
    Department of Immunotechnology, Lund University, Medicon Village (Bldg 406), 223 81 Lund, Sweden..
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity bindersInngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Surface display couples genotype with a surface exposed phenotype and thereby allows for screening of gene-encoded protein libraries for desired characteristics. Of the various display systems, phage display is by far the most popular, mainly thanks to its ability to harbor large library sizes. Here, we describe the first use of a grampositive host for display of a library of human antibody genes. The method allows for swift generation of binders by combining phage and gram-positive display, for its ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying specific low nanomolar scFv towards human HER2. The ranking and performance of the scFv isolated by flow sorting in surface immobilized form was retained when expressed as soluble scFv and analyzed by biolayer interferometry as well as after expression as full-length antibodies in mammalian cells. We also show the possibility to use gram-positive display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. We believe this combined approach has the potential for a more complete scan of the antibody repertoire and for swift affinity maturation of human antibody formats.

  • 194. Huang, Mingtao
    et al.
    Bai, Yunpeng
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. East China University of Science and Technology, China.
    Sjöström, Staffan L.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Liu, Zihe
    Petranovic, Dina
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark .
    Jönsson, Håkan N.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson Svahn, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden; Technical University of Denmark, Denmark.
    Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast2015Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 34, s. E4689-E4696Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used highthroughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed wholegenome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

  • 195.
    Hudson, Elton P.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Nikoshkov, Andrej
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 5, s. e37429-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  • 196.
    Hudson, Elton P.
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes2012Inngår i: Scientific Reports, ISSN 2045-2322, Vol. 2, s. 706-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    As antibody-based diagnosis and therapy grow at an increased pace, there is a need for methods which rapidly and accurately determine antibody-antigen interactions. Here, we report a method for the multiplex determination of antibody epitopes using bacterial cell-surface display. A protein-fragment library with 107 cell clones, covering 60 clinically-relevant protein targets, was created and characterized with massively parallel sequencing. Using this multi-target fragment library we determined simultaneously epitopes of commercial monoclonal and polyclonal antibodies targeting PSMA, EGFR, and VEGF. Off-target binding was observed for one of the antibodies, which demonstrates the method's ability to reveal cross-reactivity. We exemplify the detection of structural epitopes by mapping the therapeutic antibody Avastin. Based on our findings we suggest this method to be suitable for mapping linear and structural epitopes of monoclonal and polyclonal antibodies in a multiplex fashion and could find applicability in serum profiling as well as other protein-protein interaction studies.

  • 197. Hudson, Thomas J.
    et al.
    Anderson, Warwick
    Aretz, Axel
    Barker, Anna D.
    Bell, Cindy
    Bernabe, Rosa R.
    Bhan, M. K.
    Calvo, Fabien
    Eerola, Iiro
    Gerhard, Daniela S.
    Guttmacher, Alan
    Guyer, Mark
    Hemsley, Fiona M.
    Jennings, Jennifer L.
    Kerr, David
    Klatt, Peter
    Kolar, Patrik
    Kusuda, Jun
    Lane, David P.
    Laplace, Frank
    Lu, Youyong
    Nettekoven, Gerd
    Ozenberger, Brad
    Peterson, Jane
    Rao, T. S.
    Remacle, Jacques
    Schafer, Alan J.
    Shibata, Tatsuhiro
    Stratton, Michael R.
    Vockley, Joseph G.
    Watanabe, Koichi
    Yang, Huanming
    Yuen, Matthew M. F.
    Knoppers, M.
    Bobrow, Martin
    Cambon-Thomsen, Anne
    Dressler, Lynn G.
    Dyke, Stephanie O. M.
    Joly, Yann
    Kato, Kazuto
    Kennedy, Karen L.
    Nicolas, Pilar
    Parker, Michael J.
    Rial-Sebbag, Emmanuelle
    Romeo-Casabona, Carlos M.
    Shaw, Kenna M.
    Wallace, Susan
    Wiesner, Georgia L.
    Zeps, Nikolajs
    Lichter, Peter
    Biankin, Andrew V.
    Chabannon, Christian
    Chin, Lynda
    Clement, Bruno
    de Alava, Enrique
    Degos, Francoise
    Ferguson, Martin L.
    Geary, Peter
    Hayes, D. Neil
    Johns, Amber L.
    Nakagawa, Hidewaki
    Penny, Robert
    Piris, Miguel A.
    Sarin, Rajiv
    Scarpa, Aldo
    van de Vijver, Marc
    Futreal, P. Andrew
    Aburatani, Hiroyuki
    Bayes, Monica
    Bowtell, David D. L.
    Campbell, Peter J.
    Estivill, Xavier
    Grimmond, Sean M.
    Gut, Ivo
    Hirst, Martin
    Lopez-Otin, Carlos
    Majumder, Partha
    Marra, Marco
    Ning, Zemin
    Puente, Xose S.
    Ruan, Yijun
    Stunnenberg, Hendrik G.
    Swerdlow, Harold
    Velculescu, Victor E.
    Wilson, Richard K.
    Xue, Hong H.
    Yang, Liu
    Spellman, Paul T.
    Bader, Gary D.
    Boutros, Paul C.
    Flicek, Paul
    Getz, Gad
    Guigo, Roderic
    Guo, Guangwu
    Haussler, David
    Heath, Simon
    Hubbard, Tim J.
    Jiang, Tao
    Jones, Steven M.
    Li, Qibin
    Lopez-Bigas, Nuria
    Luo, Ruibang
    Pearson, John V.
    Quesada, Victor
    Raphael, Benjamin J.
    Sander, Chris
    Speed, Terence P.
    Stuart, Joshua M.
    Teague, Jon W.
    Totoki, Yasushi
    Tsunoda, Tatsuhiko
    Valencia, Alfonso
    Wheeler, David A.
    Wu, Honglong
    Zhao, Shancen
    Zhou, Guangyu
    Stein, Lincoln D.
    Lathrop, Mark
    Ouellette, B. F. Francis
    Thomas, Gilles
    Yoshida, Teruhiko
    Axton, Myles
    Gunter, Chris
    McPherson, John D.
    Miller, Linda J.
    Kasprzyk, Arek
    Zhang, Junjun
    Haider, Syed A.
    Wang, Jianxin
    Yung, Christina K.
    Cros, Anthony
    Liang, Yong
    Gnaneshan, Saravanamuttu
    Guberman, Jonathan
    Hsu, Jack
    Chalmers, Don R. C.
    Hasel, Karl W.
    Kaan, Terry S. H.
    Knoppers, Bartha M.
    Lowrance, William W.
    Masui, Tohru
    Rodriguez, Laura Lyman
    Vergely, Catherine
    Cloonan, Nicole
    Defazio, Anna
    Eshleman, James R.
    Etemadmoghadam, Dariush
    Gardiner, Brooke B.
    Kench, James G.
    Sutherland, Robert L.
    Tempero, Margaret A.
    Waddell, Nicola J.
    Wilson, Peter J.
    Gallinger, Steve
    Tsao, Ming-Sound
    Shaw, Patricia A.
    Petersen, Gloria M.
    Mukhopadhyay, Debabrata
    DePinho, Ronald A.
    Thayer, Sarah
    Muthuswamy, Lakshmi
    Shazand, Kamran
    Beck, Timothy
    Sam, Michelle
    Timms, Lee
    Ballin, Vanessa
    Ji, Jiafu
    Zhang, Xiuqing
    Chen, Feng
    Hu, Xueda
    Yang, Qi
    Tian, Geng
    Zhang, Lianhai
    Xing, Xiaofang
    Li, Xianghong
    Zhu, Zhenggang
    Yu, Yingyan
    Yu, Jun
    Tost, Joerg
    Brennan, Paul
    Holcatova, Ivana
    Zaridze, David
    Brazma, Alvis
    Egevad, Lars
    Prokhortchouk, Egor
    Banks, Rosamonde Elizabeth
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Viksna, Juris
    Pontén, Fredrik
    Skryabin, Konstantin
    Birney, Ewan
    Borg, Ake
    Borresen-Dale, Anne-Lise
    Caldas, Carlos
    Foekens, John A.
    Martin, Sancha
    Reis-Filho, Jorge S.
    Richardson, Andrea L.
    Sotiriou, Christos
    van't Veer, Laura
    Birnbaum, Daniel
    Blanche, Helene
    Boucher, Pascal
    Boyault, Sandrine
    Masson-Jacquemier, Jocelyne D.
    Pauporte, Iris
    Pivot, Xavier
    Vincent-Salomon, Anne
    Tabone, Eric
    Theillet, Charles
    Treilleux, Isabelle
    Bioulac-Sage, Paulette
    Decaens, Thomas
    Franco, Dominique
    Gut, Marta
    Samuel, Didier
    Zucman-Rossi, Jessica
    Eils, Roland
    Brors, Benedikt
    Korbel, Jan O.
    Korshunov, Andrey
    Landgraf, Pablo
    Lehrach, Hans
    Pfister, Stefan
    Radlwimmer, Bernhard
    Reifenberger, Guido
    Taylor, Michael D.
    von Kalle, Christof
    Majumder, Partha P.
    Pederzoli, Paolo
    Lawlor, Rita T.
    Delledonne, Massimo
    Bardelli, Alberto
    Gress, Thomas
    Klimstra, David
    Zamboni, Giuseppe
    Nakamura, Yusuke
    Miyano, Satoru
    Fujimoto, Akihiro
    Campo, Elias
    de Sanjose, Silvia
    Montserrat, Emili
    Gonzalez-Diaz, Marcos
    Jares, Pedro
    Himmelbauer, Heinz
    Bea, Silvia
    Aparicio, Samuel
    Easton, Douglas F.
    Collins, Francis S.
    Compton, Carolyn C.
    Lander, Eric S.
    Burke, Wylie
    Green, Anthony R.
    Hamilton, Stanley R.
    Kallioniemi, Olli P.
    Ley, Timothy J.
    Liu, Edison T.
    Wainwright, Brandon J.
    International network of cancer genome projects2010Inngår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 464, nr 7291, s. 993-998Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

  • 198.
    Häggmark, Anna
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Byström, Sanna
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Qundos, Ulrika
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Khademi, M.
    Olsson, T.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Antibody-based profiling of cerebrospinal fluid within multiple sclerosis2013Inngår i: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, nr 15, s. 2256-2267Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.

  • 199.
    Häggmark, Anna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mikus, Maria
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Mohsenchian, Atefeh
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hong, Mun-Gwan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Gajewska, Beata
    Baranczyk-Kuzma, Anna
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Kuzma-Kozakiewicz, Magdalena
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Plasma profiling revelas three proteins associated to amyotrophic lateral sclerosis2014Inngår i: Annals of Clinical and Translational Neurology, ISSN 2328-9503, Vol. 1, nr 8, s. 544-553Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3-5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.

    METHODS: Affinity proteomics in the form of antibody suspension bead arrays were applied to profile plasma samples from 367 ALS patients and 101 controls. The plasma protein content was directly labeled and protein profiles obtained using 352 antibodies from the Human Protein Atlas targeting 278 proteins. A focused bead array was then built to further profile eight selected protein targets in all available samples.

    RESULTS: Disease-associated significant differences were observed and replicated for profiles from antibodies targeting the proteins: neurofilament medium polypeptide (NEFM), solute carrier family 25 (SLC25A20), and regulator of G-protein signaling 18 (RGS18).

    INTERPRETATION: Upon further validation in several independent cohorts with inclusion of a broad range of other neurological disorders as controls, the alterations of these three protein profiles in plasma could potentially provide new molecular markers of disease that contribute to the quest of understanding ALS pathology.

  • 200.
    Häggmark, Anna
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Neiman, Maja
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Drobin, Kimi
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Zwahlen, Martin
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Schwenk, Jochen M
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Classification of protein profiles from antibody microarrays using heat and detergent treatment.2011Inngår i: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, nr 5, s. 564-570Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.

1234567 151 - 200 of 502
RefereraExporteraLink til resultatlisten
Permanent link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf