High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression.
A novel, cloning-independent strategy for construction of protein libraries has been developed and demonstrated experimentally. A pool of genes is prepared and thereafter extensively diluted to give one molecule of DNA per well. Each individual molecule is amplified separately by polymerase chain reaction (single-molecule PCR) yielding a PCR library. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro coupled transcription/translation. Amounts of DNA produced by the single-molecule PCR were equal and uniformity of amounts of successively in vitro synthesized proteins, which were critical for quantitative comparison among clones in the library, was better than that of the classical in vivo expression system. Here, we describe a library of anti-human serum albumin single-chain antibodies (anti-HSA-scFv) originating from a monoclonal anti-HSA-scFv which was constructed and screened in order to demonstrate its real practicability. Application of the strategy described for high-throughput generation and screening of protein libraries is discussed.
Rungpragayphan
Suang
S
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural, Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Kawarasaki
Yasuaki
Y
Imaeda
Takao
T
Kohda
Katsunori
K
Nakano
Hideo
H
Yamane
Tsuneo
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012709:Serum Albumin / Q000276:immunology; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019151:Peptide Library; D012709:Serum Albumin; D004247:DNA