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Plos One : Tailor-making a Protein A-derived Domain for Efficient Site-specific Photocoupling to Fc of Mouse Igg1, Volume 7

By Driscoll, Paul, C.

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Book Id: WPLBN0003960589
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Reproduction Date: 2015

Title: Plos One : Tailor-making a Protein A-derived Domain for Efficient Site-specific Photocoupling to Fc of Mouse Igg1, Volume 7  
Author: Driscoll, Paul, C.
Volume: Volume 7
Language: English
Subject: Journals, Science, Medical Science
Collections: Periodicals: Journal and Magazine Collection
Historic
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Publisher: Plos

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Driscoll, P. C. (n.d.). Plos One : Tailor-making a Protein A-derived Domain for Efficient Site-specific Photocoupling to Fc of Mouse Igg1, Volume 7. Retrieved from http://netlibrary.net/


Description
Description : Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG1 (mIgG1). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG1 monoclonal antibodies (mAbs). The best variant, denoted ZF5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the ZF5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG1, The best photocoupling efficiency to mIgG1 Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG1 mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG1 antibody via site-specific biotinylation, the ZF5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG1 mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the ZF5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.

 

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