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A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, no 2, 75-82 p.Article in journal (Refereed) Published
Abstract [en]

A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.

Place, publisher, year, edition, pages
2002. Vol. 35, no 2, 75-82 p.
Keyword [en]
affinity-enriched antiserum, affinity purification, expression vectors, high-throughput protein expression, immunolocalization
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-5912DOI: 10.1042/BA20010097ISI: 000174962800002PubMedID: 11916449OAI: oai:DiVA.org:kth-5912DiVA: diva2:10446
Note
QC 20100820Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Systems enabling antibody-mediated proteomics research
Open this publication in new window or tab >>Systems enabling antibody-mediated proteomics research
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels.

In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins.

A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006
Keyword
Antibody generation, dual expression, affinity purification, E. coli expression, Affibody molecules, polyclonal antibody, monospecific antibody
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-4025 (URN)91-7178-370-9 (ISBN)
Public defence
2006-06-14, Sal FD5, AlvaNova univ centrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100824Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2011-11-23Bibliographically approved

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Ståhl, Stefan

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