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  • 1.
    Berglund, Lisa
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Andrade, Jorge
    KTH, Skolan för bioteknologi (BIO).
    Odeberg, Jacob
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    The epitope space of the human proteome2008Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, nr 4, s. 606-613Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteomewide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.

  • 2.
    Emanuelsson, Olof
    et al.
    Stockholms unviersitet.
    Nielsen, H
    Stockholms universitet.
    von Heijne, G
    Stockholms universitet.
    ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites.1999Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 8, nr 5, s. 978-984Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif-finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within +/-2 residues from the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http://www.cbs.dtu.dk/services/ChloroP/.

  • 3.
    Fleetwood, Filippa
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Andersson, Ken
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Development And Optimization Of An e.Coli-based Display Platform For Selection Of Affinity Proteins2014Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, s. 135-135Artikkel i tidsskrift (Annet vitenskapelig)
  • 4.
    Hammarström, Martin
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hellgren, Niklas
    KTH, Tidigare Institutioner                               , Bioteknologi.
    van den Berg, Susanne
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.2002Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 2, s. 313-321Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.

  • 5.
    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.

  • 6. Lambert, W
    et al.
    Koeck, Philip J. B.
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Ahrman, E
    Purhonen, P
    Cheng, K
    Elmlund, D
    Hebert, Hans
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Emanuelsson, C
    Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp212011Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 2, s. 291-301Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25 degrees in relation to each other, suggesting a role for global dynamics in dodecamer function.

  • 7.
    Larsson, Per
    et al.
    Stockholm University.
    Wallner, Björn
    Stockholm University.
    Lindahl, Erik
    Stockholm University.
    Elofsson, Arne
    Stockholm University.
    Using multiple templates to improve quality of homology models in automated homology modeling2008Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 17, nr 6, s. 990-1002Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    When researchers build high-quality models of protein structure from sequence homology, it is today common to use several alternative target-template alignments. Several methods can, at least in theory, utilize information from multiple templates, and many examples of improved model quality have been reported. However, to our knowledge, thus far no study has shown that automatic inclusion of multiple alignments is guaranteed to improve models without artifacts. Here, we have carried out a systematic investigation of the potential of multiple templates to improving homology model quality. We have used test sets consisting of targets from both recent CASP experiments and a larger reference set. In addition to Modeller and Nest, a new method (Pfrag) for multiple template-based modeling is used, based on the segment-matching algorithm from Levitt's SegMod program. Our results show that all programs can produce multi-template models better than any of the single-template models, but a large part of the improvement is simply due to extension of the models. Most of the remaining improved cases were produced by Modeller. The most important factor is the existence of high-quality single-sequence input alignments. Because of the existence of models that are worse than any of the top single-template models, the average model quality does not improve significantly. However, by ranking models with a model quality assessment program such as ProQ, the average quality is improved by approximately 5% in the CASP7 test set.

  • 8.
    Lendel, Christofer
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Dincbas-Renqvist, Vildan
    KTH, Tidigare Institutioner, Bioteknologi.
    Flores, Alexander
    KTH, Tidigare Institutioner, Bioteknologi.
    Wahlberg, Elisabet
    KTH, Tidigare Institutioner, Bioteknologi.
    Dogan, Jakob
    KTH, Tidigare Institutioner, Bioteknologi.
    Nygren, Per-Åke
    KTH, Tidigare Institutioner, Bioteknologi.
    Härd, Torleif
    Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A2004Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, nr 8, s. 2078-2088Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

  • 9.
    Lindgren, Joel
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101). KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Wahlstrom, Anna
    Danielsson, Jens
    Markova, Natalia
    Ekblad, Caroline
    Graslund, Astrid
    Abrahmsen, Lars
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101). KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Warmlander, Sebastian K. T. S.
    N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity2010Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, nr 12, s. 2319-2329Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The aggregation of amyloid-beta (A beta) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease Molecules binding with high affinity and selectivity to A beta-peptides are important tools for investigating the aggregation process An A beta-binding Affibody molecule, Z(A beta 3), has earlier been selected by phage display and shown to bind A beta(1-40) with nanomolar affinity and to inhibit A beta-peptide aggregation In this study, we create truncated functional versions of the Z(A beta 3) Affibody molecule better suited for chemical synthesis production Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges The N-terminally truncated Affibody molecules Z(A beta 3)(12-58), Z(A beta 3)(15-58), and Z(A beta 3)(18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z(A beta 3)(1-58) Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z(A beta 3)(18-58), exhibited complete loss of binding to the A beta(1-40)-peptide, while the Z(A beta 3)(12-58) and Z(A beta 3)(15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the A beta(1-40)-peptide compared to the full-length Affibody molecule Nuclear magnetic resonance spectroscopy showed that the structure of A beta(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the A beta(1-40)-peptide in complex with the full-length Z(A beta 3) Affibody molecule This indicates that the N-terminally truncated Affibody molecules Z(A beta 3)(12-58) and Z(A beta 3)(15-58) are highly promising for further engineering and future use as binding agents to monomeric A beta(1-40)

  • 10. Linhult, M.
    et al.
    Binz, H. K.
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Bioteknologi.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin2002Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 2, s. 206-213Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple a-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using, circular dichroism. (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.

  • 11.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fransson, Linda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase2002Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 11, nr 6, s. 1462-1471Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, Delta(R-S)Delta(S)(divided bydivided by)., was as significant as the differential activation enthalpy, Delta(R-S)DeltaH(divided bydivided by), to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest Delta(R-S)DeltaS(divided bydivided by) 3-Hexanol displayed, contrary to other sec-alcohols, a positive Delta(R-S)DeltaS(divided bydivided by). In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MID) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the hi-her TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.

  • 12.
    Ottosson, Jenny
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Rotticci-Mulder, C
    Rotticci, D
    Hult, Karl
    KTH, Tidigare Institutioner, Bioteknologi.
    Rational design of enantio selective enzymes requires considerations of entropy2001Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 10, nr 9, s. 1769-1774Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta (R-S)DeltaH(double dagger)) and entropy (Delta (R-S)DeltaS(double dagger)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta (R-S)DeltaS(double dagger) was in all cases equally significant as Delta (R-S)DeltaH(double dagger) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger). Although the changes in Delta (R-S)DeltaH(double dagger) and Delta (R-S)DeltaS(double dagger) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta (R-S)DeltaH(double dagger). than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta (R-S)DeltaS(double dagger).

  • 13.
    Raza, Sami
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fransson, Linda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study2001Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 10, nr 2, s. 329-338Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

  • 14.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Prediction of antibody response using recombinant human protein fragments as antigen2009Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, nr 11, s. 2346-2355Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A great need exists for prediction of antibody response for the generation of antibodies toward protein targets. Earlier studies have suggested that prediction methods based on hydrophilicity propensity scale, in which the degree of exposure of the amino acid in an aqueous solvent is calculated, has limited value. Here, we show a comparative analysis based on 12,634 affinity-purified antibodies generated in a standardized manner against human recombinant protein fragments. The antibody response (yield) was measured and compared to theoretical predictions based on a large number (544) of published propensity scales. The results show that some of the scales have predictive power, although the overall Pearson correlation coefficient is relatively low (0.2) even for the best performing amino acid indices. Based on the current data set, a new propensity scale was calculated with a Pearson correlation coefficient of 0.25. The values correlated in some extent to earlier scales, including large penalty for hydrophobic and cysteine residues and high positive contribution from acidic residues, but with relatively low positive contribution from basic residues. The fraction of immunogens generating low antibody responses was reduced from 30% to around 10% if immunogens with a high propensity score (>0.48) were selected as compared to immunogens with lower scores (<0.29). The study demonstrates that a propensity scale might be useful for prediction of antibody response generated by immunization of recombinant protein fragments. The data set presented here can be used for further studies to design new prediction tools for the generation of antibodies to specific protein targets.

  • 15.
    Thul, Peter
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lindskog, Cecilia
    The human protein atlas: A spatial map of the human proteome2018Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 27, nr 1, s. 233-244Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The correct spatial distribution of proteins is vital for their function and often mis-localization or ectopic expression leads to diseases. For more than a decade, the Human Protein Atlas (HPA) has constituted a valuable tool for researchers studying protein localization and expression in human tissues and cells. The centerpiece of the HPA is its unique antibody collection for mapping the entire human proteome by immunohistochemistry and immunocytochemistry. By these approaches, more than 10 million images showing protein expression patterns at a single-cell level were generated and are publicly available at www.proteinatlas.org. The antibody-based approach is combined with transcriptomics data for an overview of global expression profiles. The present article comprehensively describes the HPA database functions and how users can utilize it for their own research as well as discusses the future path of spatial proteomics.

  • 16. Westerlund, Isabelle
    et al.
    Von Heijne, Gunnar
    Emanuelsson, Olof
    Stockholms universitet.
    LumenP - a neural network predictor for protein localization in the thylakoid lumen2003Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 12, nr 10, s. 2360-2366Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We report the development of LumenP, a new neural network-based predictor for the identification of proteins targeted to the thylakoid lumen of plant chloroplasts and prediction of their cleavage sites. When used together with the previously developed TargetP predictor, LumenP reaches a significantly better performance than what has been recorded for previous attempts at predicting thylakoid lumen location, mostly due to a lower false positive rate. The combination of TargetP and LumenP predicts around 1.5%-3% of all proteins encoded in the genomes of Arabidopsis thaliana and Oryza sativa to be located in the lumen of the thylakoid.

  • 17. Wistrand, Markus
    et al.
    Käll, Lukas
    Sonnhammer, Erik L. L.
    A general model of G protein-coupled receptor sequences and its application to detect remote homologs2006Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, nr 3, s. 509-521Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways triggered by hormones, odorants, peptides, proteins, and other types of ligands. The superfamily is so diverse that many members lack sequence similarity, although they all span the cell membrane seven times with an extracellular N and a cytosolic C terminus. We analyzed a divergent set of GPCRs and found distinct loop length patterns and differences in amino acid composition between cytosolic loops, extracellular loops, and membrane regions. We configured GPCRHMM, a hidden Markov model, to fit those features and trained it on a large dataset representing the entire superfamily. GPCRHMM was benchmarked to profile HMMs and generic transmembrane detectors on sets of known GPCRs and non-GPCRs. In a cross-validation procedure, profile HMMs produced an error rate nearly twice as high as GPCRHMM. In a sensitivity-selectivity test, GPCRHMM's sensitivity was about 15% higher than that of the best transmembrane predictors, at comparable false positive rates. We used GPCRHMM to search for novel members of the GPCR superfamily in five proteomes. All in all we detected 120 sequences that lacked annotation and are potentially novel GPCRs. Out of those 102 were found in Caenorhabditis elegans, four in human, and seven in mouse. Many predictions (65) belonged to Pfam domains of unknown function. GPCRHMM strongly rejected a family of arthropod-specific odorant receptors believed to be GPCRs. A detailed analysis showed that these sequences are indeed very different from other GPCRs. GPCRHMM is available at http://gpcrhmm.cgb.ki.se.

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