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 re...

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Bibliographic Details
Published in:Journal of molecular biology 2002-04, Vol.318 (2), p.395-405
Main Authors: Rungpragayphan, Suang, Kawarasaki, Yasuaki, Imaeda, Takao, Kohda, Katsunori, Nakano, Hideo, Yamane, Tsuneo
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal - genetics
Base Sequence
DNA - genetics
DNA Primers - genetics
Gene Amplification
Gene Expression
Genetic Techniques
Humans
in vitro coupled transcription/translation
PCR library
Peptide Library
Polymerase Chain Reaction
Protein Biosynthesis
protein library
Serum Albumin - immunology
single-chain antibody
single-molecule PCR
Transcription, Genetic
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Summary: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.
ISSN:0022-2836
1089-8638
DOI:10.1016/S0022-2836(02)00094-3