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

Mattias Brofelth, Lars Wagner Städe, Anna Isinger Ekstrand, Linn Petersson Edfeldt, Rebeka Kovačič, Thorbjørn Terndrup Nielsen, Kim Lambertsen Larsen, Laurent Duroux, Christer Wingren

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)985-996
Number of pages12
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1865
Issue number8
DOIs
Publication statusPublished - 2017 Aug

Subject classification (UKÄ)

  • Immunology in the medical area
  • Other Medical Engineering

Free keywords

  • Antibody engineering
  • Antibody microarray
  • Dock'n'Flash
  • Photochemistry
  • ScFv antibodies
  • Site-specific coupling
  • Unnatural amino acids

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