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Selection of protein epitopes for antibody production
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0002-9977-5724
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-3281-8088
2005 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 38, no 5, 723-727 p.Article in journal (Refereed) Published
Abstract [en]

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.

Place, publisher, year, edition, pages
2005. Vol. 38, no 5, 723-727 p.
Keyword [en]
Antibodies, Arrays, Computer software, Data acquisition, Enzymes, Escherichia coli, Genes, Java programming language, Optimization, Basic local alignment search tool (BLAST), Microarrays, Protein functional analysis, Transmembranes
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8827DOI: 10.2144/05385ST02ISI: 000229057100010Scopus ID: 2-s2.0-17844392695OAI: oai:DiVA.org:kth-8827DiVA: diva2:14287
Note
QC 20100907Available from: 2005-11-30 Created: 2005-11-30 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Methods for Generation and Characterization of Monospecific Antibodies
Open this publication in new window or tab >>Methods for Generation and Characterization of Monospecific Antibodies
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. xii, 66 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:20
Keyword
antibody, epitope mapping, HER2, human protein atlas, immunogenicity, proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9338 (URN)978-91-7415-139-8 (ISBN)
Public defence
2008-10-31, F3, KTH, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100907Available from: 2008-10-21 Created: 2008-10-21 Last updated: 2010-09-07Bibliographically approved

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Uhlén, MathiasSterky, Fredrik

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