SIMPLEX: Single-Molecule PCR-Linked In Vitro Expression

A Novel Method for High-Throughput Construction and Screening of Protein Libraries
  • Suang Rungpragayphan
  • Tsuneo Yamane
  • Hideo Nakano
Part of the Methods in Molecular Biology™ book series (MIMB, volume 375)


A novel strategy for construction of protein libraries called “SIMPLEX: single-molecule PCR-linked in vitro expression” is described. A pool of genes is prepared and thereafter extensively diluted to give one molecule of DNA per well. Each individual molecule is separately amplified by PCR (single-molecule PCR) yielding a one-well-one-gene PCR library. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro-coupled transcription/translation in an array format. Individual proteins in the library can be screened for target functions directly without further purification. The generated protein library is compatible with various selection methods. The strategy provides high-throughput construction and screening of protein libraries, and suits automation.

Key Words

SIMPLEX single-molecule PCR in vitro expression coupled transcription/translation PCR library protein library protein engineering high-throughput screening 


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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Suang Rungpragayphan
    • 1
  • Tsuneo Yamane
    • 2
  • Hideo Nakano
    • 3
  1. 1.Department of Biopharmacy, Faculty of PharmacySilpakorn UniversityNakorn-PathomThailand
  2. 2.Department of Environmental Biology, College of Bioscience and BiotechnologyChubu UniversityKasugaiJapan
  3. 3.Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural SciencesNagoya UniversityNagoyaJapan

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