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Targeted protein pullout from human tissue samples using competitive elution
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO), Proteomics.ORCID iD: 0000-0001-8993-048X
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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.

Place, publisher, year, edition, pages
2011. Vol. 6, no 1, 28-37 p.
Keyword [en]
Antibody, Immunoprecipitation, Methods, Proteomics, Pullout
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-14079DOI: 10.1002/biot.201000341ISI: 000287716300002Scopus ID: 2-s2.0-78651306981OAI: oai:DiVA.org:kth-14079DiVA: diva2:329595
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
In thesis
1. Affinity based proteomics research tools
Open this publication in new window or tab >>Affinity based proteomics research tools
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003.

All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:15
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11184 (URN)978-91-7415-422-1 (ISBN)
Public defence
2009-10-09, FR4 Oskar Klein, AlbaNova, Roslagstullsbacken 33, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100712Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-07-16Bibliographically approved
2. 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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