biostudies-literatureKiguchi YJapan Society for the Promotion of Science8201https://www.ebi.ac.uk/biostudies/studies/S-EPMC8050046biostudies-literatureUnknown11(1)In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two "designed" site-directed mutagenesis (A and B) targeted the N-terminal 1-10 positions of the V_H framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K_a, 3.6 × 10⁸ M^-1). Mutagenesis A substituted the amino acids at the position 1-3, 5-7, 9 and 10 with a limited set of substitutions to generate only 1,Scientific reportsThe V_H framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants.PMC805004619K07021Umezawa NOyama HKiguchi YMorita INagata YKobayashi NfalseThe V_H framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants.In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two "designed" site-directed mutagenesis (A and B) targeted the N-terminal 1-10 positions of the V_H framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K_a, 3.6 × 10⁸ M^-1). Mutagenesis A substituted the amino acids at the position 1-3, 5-7, 9 and 10 with a limited set of substitutions to generate only 1,2021-01-01T00:00:00Z2021 Apr2025-04-18T13:20:35.137Z2022-02-09T15:56:06.7ZS-EPMC80500463385925010.1038/s41598-021-87501-7