| 1. |
- Berglund, Lisa, et al.
(författare)
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The epitope space of the human proteome
- 2008
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:4, s. 606-613
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Hjelm, Barbara, et al.
(författare)
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Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
- 2011
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 20:11, s. 1824-1835
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Linhult, M., et al.
(författare)
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Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin
- 2002
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 11:2, s. 206-213
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Rockberg, Johan, et al.
(författare)
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Prediction of antibody response using recombinant human protein fragments as antigen
- 2009
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:11, s. 2346-2355
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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