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

Research output: Contribution to journalArticle


title = "Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics",
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.",
keywords = "Antibody engineering, Antibody microarray, Dock'n'Flash, Photochemistry, ScFv antibodies, Site-specific coupling, Unnatural amino acids",
author = "Mattias Brofelth and St{\"a}de, {Lars Wagner} and Ekstrand, {Anna Isinger} and {Petersson Edfeldt}, Linn and Rebeka Kovačič and Nielsen, {Thorbj{\o}rn Terndrup} and Larsen, {Kim Lambertsen} and Laurent Duroux and Christer Wingren",
year = "2017",
month = "8",
doi = "10.1016/j.bbapap.2017.03.007",
language = "English",
volume = "1865",
pages = "985--996",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "8",