Preparation of functional single-chain antibodies against bioactive gibberellins by utilizing randomly mutagenized phage-display libraries.
Screening randomly mutagenized proteins displayed on a phage surface by biopanning is a powerful strategy to obtain evolved clones with improved properties such as higher stability and functionality. We utilized this method to overcome the problem that functional single-chain antibodies against active gibberellins, a class of plant hormones, can not be prepared by some of the conventional methods. Single-chain antibody libraries with random mutations were constructed from two independent anti-bioactive gibberellin monoclonal antibody lines in a phagemid vector, so that the mutagenized scFvs were expressed in a phage-displayed form upon helper phage infection. From both libraries, scFv clones with binding activity to GA(4) were successfully obtained by successive rounds of biopanning against BSA-GA(4), the original immunogen. The results are highly suggestive that this approach might be a general solution when a single-chain antibody does not show binding activity. We found further that a ribosomal frameshift to complement a nonsense mutation frequently occurred in an amber suppressor strain of E. coli TG1, resulting in the display of a functional antibody, while such a nonsense mutant failed to produce a soluble antibody in a non-amber suppressor strain. This result explains at least partly why single-chain antibodies are sometimes functional only in a phage-displayed form, not in a soluble form.
Suzuki
Yoshihito
Y
Department of Applied Biological Chemistry, The University of Tokyo, Japan. ayoshi@pgr1.ch.a.u-tokyo.ac.jp
Ito
Shinsaku
S
Otsuka
Kaori
K
Iwasawa
Eriko
E
Nakajima
Masatoshi
M
Yamaguchi
Isomaro
I
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D005875:Gibberellins / Q000276:immunology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D005875:Gibberellins