title: Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support.
abstract: The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored 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 successfully explored the possibility to expand the genetic code of E. coli and introduced the photoreactive UAA p-benzoyl-L-phenylalanine (pBpa), and showed that the mutated scFv antibody could be expressed in E. coli with retained structural and functional properties, as well as binding affinity. The pBpa group was then used for affinity capture of the mutated antibody by β-cyclodextrin (β-CD), which provided the hydrogen atoms to be abstracted in the subsequent photocoupling process upon irradiation at 365nm. The results showed that the pBpa mutated antibody could be site-specifically photocoupled to free and surface (array) immobilized β-CD. Taken together, this paves the way for novel means of tailoring recombinant scFv antibodies for site-specific photochemical-based tagging, functionalization and immobilization in numerous applications.
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden; CREATE Health, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Linn
authors.initials: L
authors.lastname: Petersson
authors.affiliation: Dept. of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark.
authors.forename: Lars Wagner
authors.initials: LW
authors.lastname: Städe
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden; CREATE Health, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Brofelth
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Gärtner
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Elin
authors.initials: E
authors.lastname: Fors
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Sandgren
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Jacob
authors.initials: J
authors.lastname: Vallkil
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden; CREATE Health, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Niclas
authors.initials: N
authors.lastname: Olsson
authors.affiliation: Dept. of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark.
authors.forename: Kim Lambertsen
authors.initials: KL
authors.lastname: Larsen
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden; CREATE Health, Lund University, Medicon Village, SE-22381 Lund, Sweden.
authors.forename: Carl A K
authors.initials: CA
authors.lastname: Borrebaeck
authors.affiliation: Dept. of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark.
authors.forename: Laurent
authors.initials: L
authors.lastname: Duroux
authors.affiliation: Dept. of Immunotechnology, Lund University, Medicon Village, SE-22381 Lund, Sweden; CREATE Health, Lund University, Medicon Village, SE-22381 Lund, Sweden. Electronic address: christer.wingren@immun.lth.se.
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
keywords: Antibody engineering; Dock’n’flash technology; Photochemistry; Photocoupling; Protein engineering; Unnatural amino acids