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Systems enabling antibody-mediated proteomics research
KTH, School of Biotechnology (BIO).
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 [en]
Antibody generation, dual expression, affinity purification, E. coli expression, Affibody molecules, polyclonal antibody, monospecific antibody
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-4025ISBN: 91-7178-370-9 (print)OAI: oai:DiVA.org:kth-4025DiVA: diva2:10451
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
List of papers
1. A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins
Open this publication in new window or tab >>A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins
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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.

Keyword
affinity-enriched antiserum, affinity purification, expression vectors, high-throughput protein expression, immunolocalization
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-5912 (URN)10.1042/BA20010097 (DOI)000174962800002 ()11916449 (PubMedID)
Note
QC 20100820Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2010-08-23Bibliographically approved
2. A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
Open this publication in new window or tab >>A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
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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.

Keyword
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:nbn:se:kth:diva-5913 (URN)000178484900003 ()
Note
QC 20100820Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2010-08-24Bibliographically approved
3.
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4. Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
Open this publication in new window or tab >>Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1043, 33-40 p.Article in journal (Refereed) Published
Abstract [en]

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Keyword
Affinity chromatography; Antibodies; Immobilized metal-ion affinity chromatography; Protein epitope signature tags; Proteomics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5915 (URN)10.1016/j.chroma.2004.06.008 (DOI)000222728800006 ()15317410 (PubMedID)2-s2.0-3242702469 (Scopus ID)
Note
QC 20100824. 23rd International Symposium on the Separation of Proteins Peptides and Polynucleotides. Delray Beach, FL. NOV 09-12, 2003Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2011-10-18Bibliographically approved
5. Targeted protein pullout from human tissue samples using competitive elution
Open this publication in new window or tab >>Targeted protein pullout from human tissue samples using competitive elution
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2011 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 6, no 1, 28-37 p.Article in journal (Refereed) Published
Abstract [en]

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, I MAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

Keyword
Antibody, Immunoprecipitation, Methods, Proteomics, Pullout
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-14079 (URN)10.1002/biot.201000341 (DOI)000287716300002 ()2-s2.0-78651306981 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20100712 Uppdaterad från manuskript till artikel (20110316).Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2011-12-07Bibliographically approved

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