Establishing the yeast Saccharomyces cerevisiae as a system for expression of human proteins on a proteome-scale

  • Caterina Holz
  • Bianka Prinz
  • Natalia Bolotina
  • Volker Sievert
  • Konrad Büssow
  • Bernd Simon
  • Ulf Stahl
  • Christine Lang
Article

Abstract

Structural genomics requires the application of a standardised process for overexpression of soluble proteins that allows high-throughput purification and analysis of protein products. We have developed a highly parallel approach to protein expression, including the simultaneous expression screening of a large number of cDNA clones in an appropriate vector system and the use of a protease-deficient host strain. A set of 221 human genes coding for proteins of various sizes with unknown structures was selected to evaluate the system. We transferred the cDNAs from an E. coli vector to the yeast expression vector by recombinational cloning, avoiding time-consuming recloning steps and the use of restriction enzymes in the cloning process. The subcloning yield was 95%, provided that a PCR fragment of the correct size could be obtained. Sixty percent of these proteins were expressed as soluble products at detectable levels and 48% were successfully purified under native conditions using the His6 tag fusion.

The advantages of the developed yeast-based expression system are the ease of manipulation and cultivation of S. cerevisiae in the same way as with prokaryotic hosts and the ability to introduce post-translational modifications of proteins if required, thus being an attractive system for heterologous expression of mammalian proteins. The expression clones selected in this screening process are passed on to the fermentation process in order to provide milligram amounts of proteins for structure analysis within the ‘Berlin Protein Structure Factory’. All data generated is stored in a relational database and is available on our website(http://www.proteinstrukturfabrik.de).

human cDNAs recombinant fusion proteins structural genomics homologous recombination Saccharomyces cerevisiae 

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Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Caterina Holz
    • 1
    • 2
  • Bianka Prinz
    • 1
    • 2
  • Natalia Bolotina
    • 1
    • 2
  • Volker Sievert
    • 2
    • 3
  • Konrad Büssow
    • 2
    • 3
  • Bernd Simon
    • 4
  • Ulf Stahl
    • 1
  • Christine Lang
    • 1
  1. 1.Dept. Microbiology and GeneticsBerlin University of Technology, Institute for BiotechnologyBerlinGermany
  2. 2.Protein Structure FactoryBerlinGermany
  3. 3.Max-Planck Institute for Molecular GeneticsBerlinGermany
  4. 4.EMBLHeidelbergGermany

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