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A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
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2002 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, no 1, 41-50 p.Article in journal (Refereed) Published
Abstract [en]

An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

Place, publisher, year, edition, pages
2002. Vol. 99, no 1, 41-50 p.
Keyword [en]
Affibody; Affinity blotting; Affinity chromatography; cDNA; Combinatorial protein engineering; Gene expression; Proteomics; Staphylococcus aureus protein A
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-5913ISI: 000178484900003OAI: oai:DiVA.org:kth-5913DiVA: diva2:10447
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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Uhlén, MathiasNygen, Per-ÅkeStåhl, Stefan

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