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Selection of antigens for antibody-based proteomics
2008 (English)Doktorsavhandling, sammanläggning (Other scientific)
Abstract [en]

The human genome is predicted to contain ~20,500 protein-coding genes. The encoded proteins are the key players in the body, but the functions and localizations of most proteins are still unknown. Antibody-based proteomics has great potential for exploration of the protein complement of the human genome, but there are antibodies only to a very limited set of proteins. The Human Proteome Resource (HPR) project was launched in August 2003, with the aim to generate high-quality specific antibodies towards the human proteome, and to use these antibodies for large-scale protein profiling in human tissues and cells.

The goal of the work presented in this thesis was to evaluate if antigens can be selected, in a high-throughput manner, to enable generation of specific antibodies towards one protein from every human gene. A computationally intensive analysis of potential epitopes in the human proteome was performed and showed that it should be possible to find unique epitopes for most human proteins. The result from this analysis was implemented in a new web-based visualization tool for antigen selection. Predicted protein features important for antigen selection, such as transmembrane regions and signal peptides, are also displayed in the tool. The antigens used in HPR are named protein epitope signature tags (PrESTs). A genome-wide analysis combining different protein features revealed that it should be possible to select unique, 50 amino acids long PrESTs for ~80% of the human protein-coding genes.

The PrESTs are transferred from the computer to the laboratory by design of PrEST-specific PCR primers. A study of the success rate in PCR cloning of the selected fragments demonstrated the importance of controlled GC-content in the primers for specific amplification. The PrEST protein is produced in bacteria and used for immunization and subsequent affinity purification of the resulting sera to generate mono-specific antibodies. The antibodies are tested for specificity and approved antibodies are used for tissue profiling in normal and cancer tissues. A large-scale analysis of the success rates for different PrESTs in the experimental pipeline of the HPR project showed that the total success rate from PrEST selection to an approved antibody is 31%, and that this rate is dependent on PrEST length. A second PrEST on a target protein is somewhat less likely to succeed in the HPR pipeline if the first PrEST is unsuccessful, but the analysis shows that it is valuable to select several PrESTs for each protein, to enable generation of at least two antibodies, which can be used to validate each other.

Place, publisher, year, pages
Stockholm: KTH, 2008. 65 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:5
Keyword [en]
epitope, antigen, antibody, affinity, protein, proteome, proteomics, bioinformatics, prediction, primer design, sequence similarity
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4706 (URN)978-91-7178-930-3 (ISBN)oai:DiVA.org:kth-4706 (OAI)
Public defence
2008-05-09, F3, Lindstedsvägen 26, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100705Available from2008-04-22 Created:2008-04-22 Last updated:2010-09-15Bibliographically approved
List of papers
1. The epitope space of the human proteome
Open this publication in new window or tab >>The epitope space of the human proteome
2008 (English)In: Protein Science, ISSN 0961-8368, Vol. 17, no 4, 606-613Artikel i tidskrift (Refereed) Published
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.

Keyword
proteomics; antigen; epitope; sequence similarity; antibody; grid; B-CELL EPITOPES; ANTIGENIC DETERMINANTS; DATA-BANK; PREDICTION; ANTIBODIES; MYOGLOBIN; PEPTIDES; SEQUENCE; LIBRARIES; PROTEINS
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8255 (URN)10.1110/ps.073347208 (DOI)000254197800002 (ISI)2-s2.0-41649104373 (ScopusID)
Note
QC 20100705Available from2008-04-22 Created:2008-04-22 Last updated:2010-07-05Bibliographically approved
2. A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation
Open this publication in new window or tab >>A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation
Show others...
2008 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 14, 2832-2839Artikel i tidskrift (Refereed) Published
Abstract [en]

Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.

Keyword
antibody generation, antigen selection, bioinformatics, epitope, sequence similarity, CELL EPITOPE PREDICTION, HUMAN PROTEOME, B-CELL, DETERMINANTS, EXPRESSION, PROTEINS, PROGRAM, SITES, ATLAS, GENE
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8256 (URN)10.1002/pmic.200800203 (DOI)000258117400007 (ISI)2-s2.0-48949107645 (ScopusID)
Note
QC 20100705Available from2008-04-22 Created:2008-04-22 Last updated:2010-09-07Bibliographically approved
3. Primer design for high-throughput PCR cloning
Open this publication in new window or tab >>Primer design for high-throughput PCR cloning
(English)In: Artikel i tidskrift (Other academic) Submitted
Keyword
primer design, RT-PCR, cloning
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8257 (URN)
Note
QC 20100705Available from2008-04-22 Created:2008-04-22 Last updated:2010-07-05Bibliographically approved
4. Generation of validated antibodies towards the human proteome
Open this publication in new window or tab >>Generation of validated antibodies towards the human proteome
Show others...
(English)In: Artikel i tidskrift (Other academic) Submitted
Abstract [en]

Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8258 (URN)
Note
QC 20100705Available from2008-04-22 Created:2008-04-22 Last updated:2010-07-05Bibliographically approved

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