The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants
Abstract 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...
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2021-04-01
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Online Access: | https://doi.org/10.1038/s41598-021-87501-7 |
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doaj-8bb6256cd6294d32b16c979a6b21bdcb2021-04-18T11:35:38ZengNature Publishing GroupScientific Reports2045-23222021-04-0111111110.1038/s41598-021-87501-7The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutantsYuki Kiguchi0Hiroyuki Oyama1Izumi Morita2Yasuhiro Nagata3Naoko Umezawa4Norihiro Kobayashi5Kobe Pharmaceutical UniversityKobe Pharmaceutical UniversityKobe Pharmaceutical UniversityKobe Pharmaceutical UniversityKobe Pharmaceutical UniversityKobe Pharmaceutical UniversityAbstract 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 VH 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 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1 K a values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest K a value, 1.1 × 1010 M−1.https://doi.org/10.1038/s41598-021-87501-7 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi |
spellingShingle |
Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants Scientific Reports |
author_facet |
Yuki Kiguchi Hiroyuki Oyama Izumi Morita Yasuhiro Nagata Naoko Umezawa Norihiro Kobayashi |
author_sort |
Yuki Kiguchi |
title |
The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants |
title_short |
The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants |
title_full |
The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants |
title_fullStr |
The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants |
title_full_unstemmed |
The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants |
title_sort |
vh framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scfv mutants |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2021-04-01 |
description |
Abstract 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 VH 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 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1 K a values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest K a value, 1.1 × 1010 M−1. |
url |
https://doi.org/10.1038/s41598-021-87501-7 |
work_keys_str_mv |
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