Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics

Research output: Contribution to journalArticle

Abstract

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

Details

Authors
Organisations
External organisations
  • Aalborg University
  • Lund University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area
  • Other Medical Engineering

Keywords

  • Antibody engineering, Antibody microarray, Dock'n'Flash, Photochemistry, ScFv antibodies, Site-specific coupling, Unnatural amino acids
Original languageEnglish
Pages (from-to)985-996
Number of pages12
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1865
Issue number8
Publication statusPublished - 2017 Aug
Publication categoryResearch
Peer-reviewedYes

Related research output

Mattias Brofelth, 2018 Jan 30, Lund: Department of Immunotechnology, Lund University. 147 p.

Research output: ThesisDoctoral Thesis (compilation)

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