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Generation of validated antibodies towards the human proteome
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
Uppsala Univ, Rudbeck laboratory.
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(Engelska)Artikel i tidskrift (Övrigt vetenskapligt) 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.

Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-8258OAI: oai:DiVA.org:kth-8258DiVA, id: diva2:13532
Anmärkning
QC 20100705Tillgänglig från: 2008-04-22 Skapad: 2008-04-22 Senast uppdaterad: 2010-07-05Bibliografiskt granskad
Ingår i avhandling
1. Selection of antigens for antibody-based proteomics
Öppna denna publikation i ny flik eller fönster >>Selection of antigens for antibody-based proteomics
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2008. s. 65
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:5
Nyckelord
epitope, antigen, antibody, affinity, protein, proteome, proteomics, bioinformatics, prediction, primer design, sequence similarity
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-4706 (URN)978-91-7178-930-3 (ISBN)
Disputation
2008-05-09, F3, Lindstedsvägen 26, Stockholm, 10:00
Opponent
Handledare
Anmärkning
QC 20100705Tillgänglig från: 2008-04-22 Skapad: 2008-04-22 Senast uppdaterad: 2010-09-15Bibliografiskt granskad

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Berglund, LisaBjörling, ErikGry, MarcusAl-Khalili Szigyarto, CristinaPersson, AnjaOttoson, JennyWernérus, HenrikNilsson, PeterSivertsson, ÅsaHober, SophiaUhlén, Mathias
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