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Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles

Authors

  • Yuan Lu,

    1. Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025; telephone: 650-723-5398; fax: 650-725-0555
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  • John P. Welsh,

    1. Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025; telephone: 650-723-5398; fax: 650-725-0555
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  • Wei Chan,

    1. Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025; telephone: 650-723-5398; fax: 650-725-0555
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  • James R. Swartz

    Corresponding author
    1. Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025; telephone: 650-723-5398; fax: 650-725-0555
    2. Department of Bioengineering, Stanford University, Stanford, California 94305
    • Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025; telephone: 650-723-5398; fax: 650-725-0555
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Abstract

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold. Biotechnol. Bioeng. 2013; 110: 2073–2085. © 2013 Wiley Periodicals, Inc.

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