Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.
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.
Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
Mattias
M
Brofelth
Dept. of Chemistry and Bioscience, Aalborg University, DK-92200 Aalborg, Denmark.
Lars Wagner
LW
Städe
Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
Anna Isinger
AI
Ekstrand
Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
Linn Petersson
LP
Edfeldt
Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
Rebeka
R
Kovačič
Dept. of Chemistry and Bioscience, Aalborg University, DK-92200 Aalborg, Denmark.
Thorbjørn Terndrup
TT
Nielsen
Dept. of Chemistry and Bioscience, Aalborg University, DK-92200 Aalborg, Denmark.
Kim Lambertsen
KL
Larsen
Dept. of Chemistry and Bioscience, Aalborg University, DK-92200 Aalborg, Denmark.
Laurent
L
Duroux
Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.. Electronic address: christer.wingren@immun.lth.se.
Christer
C
Wingren
D000915:Antibody Affinity / Q000276:immunology*; D001577:Benzophenones / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D009154:Mutation / Q000235:genetics* / Q000276:immunology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000276:immunology / Q000378:metabolism
C488060:4-benzoylphenylalanine; D001577:Benzophenones; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D047392:beta-Cyclodextrins; D010649:Phenylalanine; C031215:betadex
Antibody engineering; Antibody microarray; Dock'n'Flash; Photochemistry; Site-specific coupling; Unnatural amino acids; scFv antibodies