Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Authors

  • Zhi-Gang Qian,

    1. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea; telephone: +82-42-869-3930; fax: +82-42-869-3910
    Search for more papers by this author
  • Xiao-Xia Xia,

    1. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea; telephone: +82-42-869-3930; fax: +82-42-869-3910
    Search for more papers by this author
  • Jong Hyun Choi,

    1. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea; telephone: +82-42-869-3930; fax: +82-42-869-3910
    Search for more papers by this author
  • Sang Yup Lee

    Corresponding author
    1. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea; telephone: +82-42-869-3930; fax: +82-42-869-3910
    2. Department of Bio and Brain Engineering, and Bioinformatics Research Center, KAIST, Daejeon, Republic of Korea
    • Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea; telephone: +82-42-869-3930; fax: +82-42-869-3910.
    Search for more papers by this author

  • Zhi-Gang Qian and Xiao-Xia Xia are contributed equally to this work.

Abstract

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis α-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250–700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins. Biotechnol. Bioeng. 2008;101: 587–601. © 2008 Wiley Periodicals, Inc.

Ancillary