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Recombinant Gene Expression

Volume 267 of the series Methods in Molecular Biology pp 267-276

High-Throughput Expression in Microplate Format in Saccharomyces cerevisiae

  • Caterina HolzAffiliated withBerlin Technical University, Institute for Biotechnology
  • , Christine LangAffiliated withBerlin Technical University, Institute for Biotechnology

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Abstract

We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.

Key Words

Saccharomyces cerevisiae microplate format CUP1 promoter epitope tagging