Change search
Refine search result
12 1 - 50 of 59
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the 'Create feeds' function.
  • 1.
    Akpe, Victor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Characterization studies of aluminum phthalocyanine binding to antibodies from SKBR 3 cell line2008Report (Other academic)
  • 2. Alkharusi, Amira
    et al.
    Yu, Shengze
    KTH, School of Biotechnology (BIO).
    Landazuri, Natalia
    Zadjali, Fahad
    Davodi, Belghis
    Nystrom, Thomas
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Rahbar, Afsar
    Norstedt, Gunnar
    Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 48, 79558-79569 p.Article in journal (Refereed)
    Abstract [en]

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

  • 3. Altai, M.
    et al.
    Liu, H.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, A.
    Tolmachev, V.
    Gräslund, T.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Improving of molecular design of a novel Affibody-fused HER2-recognising anticancer toxin using radionuclide-based techniques2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, S178-S178 p.Article in journal (Refereed)
  • 4. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, School of Biotechnology (BIO), Protein Technology.
    Orlova, Anna
    Tolmachev, Vladimir
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin2016In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 3, 1185-1194 p.Article in journal (Refereed)
    Abstract [en]

    Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide In-111. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)(3) purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, In-111-(HE)(3)-Z(HER2)-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.

  • 5. Danielsen, S.
    et al.
    Eklund, M.
    Deussen, H. J.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Borchert, T. V.
    In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor2001In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 272, no 02-jan, 267-274 p.Article in journal (Refereed)
    Abstract [en]

    The 'detergent lipase' Lipolasel((R)), from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase((R)) could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase((R)) could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase((R))-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase((R)) variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.

  • 6. Elgstrand Wettergren, E.
    et al.
    Seijsing, Johan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Sest, M.
    Quintino, L.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lundberg, C.
    Up-regulation of endogenous GAD67 expression in vivo using designed zinc finger-based transcription factorsManuscript (preprint) (Other academic)
  • 7. Gordley, Russll M.
    et al.
    Smith, Justin D.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Barbas, Carlos F., III
    Evolution of programmable zinc finger-recombinases with activity in human cells2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 367, no 3, 802-813 p.Article in journal (Refereed)
    Abstract [en]

    Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (ReCZFS) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites, One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in similar to 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)S may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.

  • 8.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO).
    Shibasaki, Seiji
    KTH, School of Biotechnology (BIO).
    Vernet, Erik
    KTH, School of Biotechnology (BIO).
    Skogs, Marie
    KTH, School of Biotechnology (BIO).
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Selection and characterization of affibody molecules interfering with the interaction between Ras and RafManuscript (Other academic)
  • 9.
    Grimm, Sebastian
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Yu, Feifan
    KTH, School of Biotechnology (BIO), Proteomics.
    Shibasaki, Seiji
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro2010In: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, no 6, 766-773 p.Article in journal (Refereed)
    Abstract [en]

    Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

  • 10.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Affibody molecules: therapy and in vivo diagnostic applications2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, S48-S48 p.Article in journal (Refereed)
  • 11.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Affibody molecules: Therapy and in vivo diagnostic applications2015In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 36, S98-S98 p.Article in journal (Other academic)
  • 12.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Protein Engineering by Directed Evolution and Rational Design2001Doctoral thesis, comprehensive summary (Other scientific)
  • 13.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Ehn, Maria
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Gunnel, Lundin
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner2002In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 942, no 1-2, 157-166 p.Article in journal (Refereed)
    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.

  • 14.
    Gräslund, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Eriksson, Tove
    Jonsson, Andreas
    Bergman, Thomas
    Li, Jingjing
    Lendel, Christofer
    Jarstad, Anders
    IGF-1R binding polypeptides and their use2007Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    This invention relates to polypeptides which bind to IGF-1R and to applications of those polypeptides in medicine, veterinary medicine, diagnostics and imaging.

  • 15.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hedhammar, My
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 96, no 1, 93-102 p.Article in journal (Refereed)
    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.

  • 16.
    Gräslund, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Li, X. L.
    Magnenat, L.
    Popkov, M.
    Barbas, C. F.
    Exploring strategies for the design of artificial transcription factors2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 5, 3707-3714 p.Article in journal (Refereed)
    Abstract [en]

    Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be, in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of gamma-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the -117-position of the gamma-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, ggl-VP64-RA. This transcription factor was able to interact directly with the gamma-globin promoter and up-regulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-RA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The gamma-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.

  • 17.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lundin, Gunnel
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Charge engineering of a protein domain to allow efficient ion-exchange recovery2000In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, 703-709 p.Article in journal (Refereed)
    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.

  • 18.
    Gräslund, Torbjörn
    et al.
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nilsson, Joakim
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Lindberg, Michael
    Uhlén, Mathias
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Production of a Thermostable DNA Polymerase by Site-Specific Cleavage of a Heat-Eluted Affinity Fusion Protein1997In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 9, 125-132 p.Article in journal (Refereed)
    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.

  • 19. Gunaydin, Gokce
    et al.
    Yu, Shengze
    KTH, School of Biotechnology (BIO), Protein Technology.
    Graslund, Torbjorn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Hammarstrom, Lennart
    Marcotte, Harold
    Fusion of the mouse IgG1 Fc domain to the VHH fragment (ARP1) enhances protection in a mouse model of rotavirus2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 30171Article in journal (Refereed)
  • 20. Hedhammar, M.
    et al.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Protein engineering strategies for selective protein purification2005In: Chemical Engineering & Technology, ISSN 0930-7516, E-ISSN 1521-4125, Vol. 28, no 11, 1315-1325 p.Article, review/survey (Refereed)
    Abstract [en]

    When producing and purifying recombinant proteins it is of importance to minimize the number of unit operations during the purification procedure. This is accomplished by increasing the selectivity in each step. Due to the high selectivity of affinity chromatography it has a widespread use in protein purification. However, most target proteins lack a suitable affinity ligand usable for capture oil a solid matrix. A way to circumvent this obstacle is to genetically fuse the gene encoding the target protein with a gene encoding a purification tag. When the chimeric protein is expressed, the tag allows for specific capture of the fusion protein. In industrial-scale production, extension of the target protein often is unwanted since it might interfere with the function of the target protein. Hence, a purification scheme developed for the native protein is desired. In this review, different fusion strategies used for protein purification are discussed. Also, the development of ligands for selective affinity purification of native target proteins is surveyed.

  • 21.
    Hedhammar, My
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Alm, Tove
    KTH, School of Biotechnology (BIO), Proteomics.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag2006In: Biotechnology Journal, ISSN 1860-6768, Vol. 1, 187-196 p.Article in journal (Refereed)
    Abstract [en]

    A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

  • 22.
    Hedhammar, My
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Negatively charged purification tags for selective anion-exchange recovery2004In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 11, 779-786 p.Article in journal (Refereed)
    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.

  • 23.
    Hofström, Camilla
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Altai, Mohamed
    Honarvar, H.
    Strand, J.
    Malmberg, J.
    Hosseinimehr, Seyed Jalal
    Orlova, Anna
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    HAHAHA, HEHEHE,HIHIHI or HKHKHK: influence of position and composition of histidine containing tags on biodistribution of [99mTc(CO)3]+-labeled Affibody moleculesManuscript (preprint) (Other academic)
  • 24.
    Hofström, Camilla
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Altai, Mohamed
    Honarvar, Hadis
    Strand, Joanna
    Malmberg, Jennie
    Hosseinimehr, Seyed Jalal
    Orlova, Anna
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    HAHAHA, HEHEHE, HIHIHI, or HKHKHK: Influence of Position and Composition of Histidine Containing Tags on Biodistribution of [Tc-99m(CO)(3)](+)-Labeled Affibody Molecules2013In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 56, no 12, 4966-4974 p.Article in journal (Refereed)
    Abstract [en]

    Engineered affibody molecules can be used for high contrast in vivo molecular imaging. Extending a recombinantly produced HER2 binding affibody molecule with a hexa-histidine tag allows for convenient purification by immobilized metal-ion affinity chromatography and labeling with [Tc-99m(CO)(3)](+) but increases radioactivity uptake in the liver. To investigate the impact of charge, lipophilicity, and position on biodistribution, 10 variants of a histidine-based tag was attached to a HER2 binding affibody molecule. The biochemical properties and the HER2 binding affinity appeared to be similar for all variants. In vivo, positive charge promoted liver uptake. For N-terminally placed tags, promoted liver uptake and decreased kidney uptake. Kidney uptake was higher for C-terminally placed tags compared to their N-terminal counterparts. The variant with the amino acid composition HEHEHE placed in the N-terminus gave the lowest nonspecific uptake.

  • 25.
    Hofström, Camilla
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Orlova, Anna
    Altai, Mohamed
    Wångsell, Fredrik
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, Vladimir
    Use of a HEHEHE Purification Tag Instead of a Hexahistidine Tag Improves Biodistribution of Affibody Molecules Site-Specifically Labeled with Tc-99m, In-111 and I-1252011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 11, 3817-3826 p.Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of small (similar to 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging in vivo. The attachment of a hexahistidine (His(6))-tag to the Affibody molecule allows facile purification by immobilized metal ion affinity chromatography (IMAC) but leads to high accumulation of radioactivity in the liver. Earlier, we have demonstrated that replacement of the His(6)- tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of Affibody molecules by IMAC, enables labeling with [Tc-99m(CO)(3)](+), and provides low hepatic accumulation of radioactivity. In this study, we compared the biodistribution of cysteine-containing Affibody molecules site-specifically labeled with In-111, Tc-99m, and I-125 at the C-terminus, having a His(6)-tag at the N- or C-terminus or a HEHEHE-tag at the N-terminus. We show that the use of a HEHEHE-tag provides appreciable reduction of hepatic radioactivity, especially for radiometal labels. We hope that this information can also be useful for development of other scaffold protein-based imaging agents.

  • 26.
    Hofström, Camilla
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Stadler, C.
    Vermet, E.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Step-wise down regulationof the epidermal growth factor receptor by affinity-based intracellular redirectionManuscript (preprint) (Other academic)
  • 27.
    Larsson, Karin
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Lindskog, C.
    Hansson, M.
    Angelidou, P.
    Hökfelt, T.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Wernérus, H.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101). KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Novel antigen design for the generation of antibodies to G-protein-coupled receptors2011In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 370, no 1-2, 14-23 p.Article in journal (Refereed)
    Abstract [en]

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

  • 28.
    Larsson, Karin
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO).
    Lindskog, C
    Uhlén, Mattias
    KTH, School of Biotechnology (BIO), Proteomics.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Hansson, M
    Hober, Sophia
    KTH, School of Biotechnology (BIO).
    Angelidou, Pia
    KTH, School of Biotechnology (BIO).
    Hökfelt, T
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO).
    Novel antigen design for the generation of G-protein-coupled receptorantibodies.Manuscript (preprint) (Other academic)
  • 29. Legendre, Daniel
    et al.
    Laraki, Nezha
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Björnvad, Mads E
    Bouchet, Michèle
    Nygren, Per-Åke
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Borchert, Torben V
    Fastrez, Jacques
    Display of active subtilisin 309 on phage: Analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors2000In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 87-102 p.Article in journal (Refereed)
    Abstract [en]

    Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial Libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close Link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentils on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very Limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-L-Ala-L-Ala-L-Pro- Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-L-Lys-L-Ala- L-Pro-Phe(P)-diphenylester ter allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

  • 30. Lewensohn, Rolf
    et al.
    Orre, Lukas
    Lehtiö, Janne
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    S100a6 and/or s100a4 inhibitors for treating cancer2008Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    Aspects of this invention relate to the fields of molecular biology and medicine. More specifically, disclosed herein are several approaches to provide subjects suffering from cancer with an inhibitor of S100A6 and/or S 100A4 alone or in combination with other cancer therapies so as to improve the cancer therapy and/or more efficiently treat cancer, in particular forms of cancer that are resistant to other therapies. Also disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for metastases. Further, disclosed herein are approaches for using S 100A6 and/or S 100A4 as a biomarker for cancer therapies, in particular, as a biomarker to determine individual responses to cancer therapies. In addition, disclosed herein are approaches to identifying S 100A6 and/or S 100A4 inhibitors, for example, that act synergistically with a cancer therapy.

  • 31.
    Li, Jingjing
    et al.
    KTH, School of Biotechnology (BIO).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO).
    Vernet, Erik
    KTH, School of Biotechnology (BIO).
    Höidén-Guthenberg, Ingmarie
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Selection of affibody molecules blocking hormone-binding to the insulin-like growth factor 1 receptorManuscript (Other academic)
  • 32.
    Li, Jingjing
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Larsson, Barbro
    Höidén-Guthenberg, Ingmarie
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection of affibody molecules to the ligand-binding site of the insulin-like growth factor-1 receptor2010In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 55, 99-109 p.Article in journal (Refereed)
    Abstract [en]

    Affibody molecules binding to the site of hormone interaction in IGF-IR (insulin-like growth factor-I receptor) were successfully selected by phage-display technology employing a competitive-elution strategy during biopanning, whereby release of receptor-bound phagemids was accomplished by competition with IGFI (insulin-like growth factor-I). In non-competitive selections, the elution of receptor-bound phagemids was performed by imidazole or low-pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA-based assay showed that the affibody molecules generated by IGF-I competition during elution, in addition to affibody molecules generated in the noncompetitive selections, could compete with IGF-I for binding to the receptor. The affinities of the isolated variants to IGF-IR-overexpressing MCF-7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z(4;40), was shown to recognize IGF- IR efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull-down from cell extracts. In addition, when Z, was added to the medium of MCF-7 cells that were dependent on IGF-I for efficient growth, it was found to have a dose-dependent growth-inhibitory effect on the cells. Applications of affibody-based reagents for quantitative and qualitative analyses of IGF- I R status, as well as applications of affibody-based reagents for therapy, are discussed.

  • 33.
    Lindberg, Hanna
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Altai, Mohamed
    Honorvar, Hadis
    Wållberg, Helena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Orlova, Anna
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Tolmachev, Vladimir
    Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for Tc-99m-labelling at the C terminus2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no 3, 641-651 p.Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide Tc-99m. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with Tc-99m. Tc-99m-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, Tc-99m-Z(HER2:2395)-VDC and Tc-99m-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of Tc-99m-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of Tc-99m-Z(HER2:2395)-VDC, but it was substantially higher than uptake of Tc-99m-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of Tc-99m was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of Tc-99m labelling due to a partial loss of site-specificity of nuclide chelation.

  • 34. Linhult, M.
    et al.
    Gulich, S.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand2003In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 16, no 12, 1147-1152 p.Article in journal (Refereed)
    Abstract [en]

    Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD* dimer. The ABD* dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution ( pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.

  • 35. Linhult, M.
    et al.
    Gulich, S.
    Gräslund, Torbjörn
    KTH, Superseded Departments, Biotechnology.
    Simon, A.
    Karlsson, M.
    Sjoberg, A.
    Nord, K.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach2004In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 55, no 2, 407-416 p.Article in journal (Refereed)
    Abstract [en]

    Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its nonengineered form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.

  • 36.
    Liu, Hao
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Seijsing, Johan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik Y.
    Tolmachev, Vladimir
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z(HER2:2891)-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain2015In: International Journal of Oncology, ISSN 1019-6439, Vol. 47, no 2, 601-609 p.Article in journal (Refereed)
    Abstract [en]

    Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (K-D=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (K-D=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

  • 37.
    Lundberg, Emma
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Selection and characterization of Affibody (R) ligands to the transcription factor c-Jun2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, 17-27 p.Article in journal (Refereed)
    Abstract [en]

    c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody (R) ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold protein. Homodimeric c-Jun protein was recombinantly produced in Escherichia coli and, prior to selection, the quality of the target protein was investigated by binding analyses, which indicated specific binding to a double-stranded DNA hairpin construct containing a c-Jun response element, but not to a control sequence. Isolated Affibody (R) variants from the phage selection were expressed in E. coli, purified by affinity chromatography and their interaction with c-Jun was analysed. In biosensor analyses, one Affibody (R) ligand, denoted Z(cJun518) was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 mu M. By constructing a head-to-tail homodimeric version of Z(cJun518), its apparent affinity for c-Jun could be increased threefold, suggesting co-operativity effects in the binding to the immobilized c-Jun protein. Further characterization of the Z(cJun518) Affibody (R) molecule demonstrated, in both affinity-capture and Western-blotting experiments, its ability to interact selectively with c-Jun, even when the c-Jun target was present in a complex protein background consisting of a bacterial cell lysate. Z(cJun518) could also be used to stain the c-Jun-overexpressing cell line C8161 visualized by confocal fluorescence microscopy. Results from competition experiments indicated that the binding epitope on c-Jun for the Z(cJun518) Affibody (R) molecule was separate from the binding sites of both a polyclonal antibody raised against the unstructured N-terminal domain and a double-stranded DNA hairpin containing a c-Jun response element. The potential intracellular use of Affibody (R) ligands directed against transcription factors and other oncogenic factors is discussed.

  • 38.
    Lundberg, Emma
    et al.
    KTH, School of Biotechnology (BIO).
    Höidén-Guthenberg, Ingmarie
    Affibody AB.
    Larsson, Barbro
    Affibody AB.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Site-specifically conjugated anti-HER2 Affibody® molecules as one-step reagents for target expression analyses on cells and xenograft samples2007In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 319, no 1-2, 53-63 p.Article in journal (Refereed)
    Abstract [en]

    Affibody (R) molecules are a class of small and robust affinity proteins that can be generated to interact with a variety of antigens, thus having the potential to provide useful tools for biotechnological research and diagnostic applications. In this study, we have investigated Affibody-based reagents interacting specifically with the tyrosine kinase receptor HER2. A head-to-tail dimeric construct was site-specifically conjugated with different fluorescent and enzymatic groups resulting in reagents that were used for detection and quantification. The amount of cell surface expressed HER2 oil eleven (11) well characterized cell lines was quantified relative to each other by flow cytometry and shown to correlate well with results from parallel analyses of HER2 mRNA levels measured by real-time PCR. Further, immunofluorescence I microscopy studies of the cell lines and immunohistochemical analyses of cryosections of HER2 expressing SKOV-3 xenografts showed strong staining of the plasma membrane of tumor cells with little background staining. Full-length HER2 protein Could also be efficiently recovered from a cell extract by an immunoprecipitation procedure, using an Affibody ligand-based resin.

    These novel non-IgG derived reagents could be used to detect and quantify HER2 expression. By adapting the methods for use with Affibody molecules binding to other cell surface receptors, it is anticipated that also these receptors can be detected and quantified in a similar manner.

  • 39.
    Lundberg, Emma
    et al.
    KTH, School of Biotechnology (BIO).
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO).
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase2007In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 322, no 1-2, 40-49 p.Article in journal (Refereed)
    Abstract [en]

    Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for throughput applications.

  • 40. Mitran, B.
    et al.
    Altai, M.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, H.
    Widstrom, C.
    Orlova, A.
    Tolmachev, V.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Evaluation of 99mTc-ZIGF1R: 4551-GGGC Affibody Molecule, a New Construct for Imaging the Insulin-like Growth Factor Type 1 Receptor Expression2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, S440-S440 p.Article in journal (Other academic)
  • 41. Mitran, Bogdan
    et al.
    Altai, Mohamed
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Protein Technology.
    Honarvar, Hadis
    Sandström, Mattias
    Orlova, Anna
    Tolmachev, Vladimir
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Evaluation of Tc-99m-Z(IGF1R:4551)-GGGC affibody molecule, a new probe for imaging of insulin-like growth factor type 1 receptor expression2015In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 47, no 2, 303-315 p.Article in journal (Refereed)
    Abstract [en]

    Overexpression of insulin-like growth factor-1 receptor (IGF-1R) in several cancers is associated with resistance to therapy. Radionuclide molecular imaging of IGF-1R expression in tumors may help in selecting the patients that will potentially respond to IGF-1R-targeted therapy. Affibody molecules are small (7 kDa) non-immunoglobulin-based scaffold proteins that are well-suited probes for radionuclide imaging. The aim of this study was the evaluation of an anti-IGF-1R affibody molecule labeled with technetium-99m using cysteine-containing peptide-based chelator GGGC at C-terminus. Z(IGF1R:4551)-GGGC was efficiently and stably labeled with technetium-99m (radiochemical yield 97 +/- A 3 %). Tc-99m-Z(IGF1R:4551)-GGGC demonstrated specific binding to IGF-1R-expressing DU-145 (prostate cancer) and MCF-7 (breast cancer) cell lines and slow internalization in vitro. The tumor-targeting properties were studied in BALB/c nu/nu mice bearing DU-145 and MCF-7 xenografts. [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was used for comparison. The biodistribution study demonstrated high tumor-to-blood ratios (6.2 +/- A 0.9 and 6.9 +/- A 1.0, for DU-145 and MCF-7, respectively, at 4 h after injection). Renal radioactivity concentration was 16-fold lower for Tc-99m-Z(IGF1R:4551)-GGGC than for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) at 4 h after injection. However, the liver uptake of Tc-99m-Z(IGF1R:4551)-GGGC was 1.2- to 2-fold higher in comparison with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551). A possible reason for the elevated hepatic uptake of Tc-99m-Z(IGF1R:4551)-GGGC is a high lipophilicity of amino acids in the binding site of Z(IGF1R:4551), which is not compensated in Tc-99m-Z(IGF1R:4551)-GGGC. In conclusion, Tc-99m-Z(IGF1R:4551)-GGGC can visualize the IGF-1R expression in human tumor xenografts and provides low retention of radioactivity in kidneys. Further development of this imaging agent should include molecular design aimed at reducing the hepatic uptake.

  • 42. Orlova, A.
    et al.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Malmberg, J.
    Ahlgren, S.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Tolmachev, V.
    HEHEHE: a new chelator for [Tc-99m(CO)(3)](+)-labeling assembling His(6)-tag in protein purification2010In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no Suppl. 2, S264-S265 p.Article in journal (Other academic)
  • 43. Orlova, A.
    et al.
    Malmberg, J.
    Hofström, Camilla
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Abrahmsen, L.
    Bergman, T.
    Sjöberg, A.
    Sandström, M.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Tolmachev, V.
    Affibody molecule 111In-DOTA-ZIGF1R:4551, a new potential probe for imaging of IGF-1R expression in malignant tumours2011In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 38, S171-S171 p.Article in journal (Other academic)
  • 44. Orre, L. M.
    et al.
    Panizza, E.
    Kaminskyy, V. O.
    Vernet, Erik
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Zhivotovsky, B.
    Lehtiö, J.
    S100A4 interacts with p53 in the nucleus and promotes p53 degradation2013In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 49, 5531-5540 p.Article in journal (Refereed)
    Abstract [en]

    S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.

  • 45. Orre, Lukas
    et al.
    Panizza, Elena
    Kaminskyy, Vitaliy
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Zhivotovsky, Boris
    Lehtiö, Janne
    S100A4 interacts with p53 in the nucleus promoting its degradation2013In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 73, no 8Article in journal (Other academic)
  • 46.
    Qin, Haiyan
    et al.
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Quantum dot-affibody fluorescence probes for single molecule tracking in living cell membranesManuscript (preprint) (Other academic)
  • 47.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Johansson, P.
    Eklund, P.
    Höidén-Guthenberg, I.
    Frejd, F.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Reduction of immunoglobulin G in mice by an affibody molecule blocking the interaction between immunoglobulin G and the neonatal Fc receptorManuscript (preprint) (Other academic)
  • 48.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Höidén-Guthenberg, I.
    Bönisch, H.
    Gunneriusson, E.
    Frejd, F.
    Abramsén, L.
    Ekblad, C.
    Löfblom, J.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    An engineered affibody molecule with pHdependent binding to FcRn mediates extended circulatory half-lifeof a fusion proteinManuscript (preprint) (Other academic)
  • 49.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Höidén-Guthenberg, Ingmarie
    Bönisch, Heiko
    Guneriusson, Elin
    Frejd, Fredrik Y.
    Abrahmsén, Lars
    Ekblad, Caroline
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    An engineered affibody molecule with pH-dependent binding to FcRn mediates extended circulatory half-life of a fusion protein2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 48, 17110-17115 p.Article in journal (Refereed)
    Abstract [en]

    Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased halflife, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.

  • 50.
    Seijsing, Johan
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindborg, Malin
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Gräslund, Torbjörn
    KTH, School of Biotechnology (BIO), Protein Technology.
    Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, e81350- p.Article in journal (Refereed)
    Abstract [en]

    Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

12 1 - 50 of 59
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf