Cancer Institute A national cancer institute
designated cancer center

James Swartz

Publication Details

  • Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles BIOTECHNOLOGY AND BIOENGINEERING Lu, Y., Welsh, J. P., Chan, W., Swartz, J. R. 2013; 110 (8): 2073-2085


    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;9999: XX-XX. © 2013 Wiley Periodicals, Inc.

    View details for DOI 10.1002/bit.24903

    View details for Web of Science ID 000320930800002

    View details for PubMedID 23519642

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