4.6 Article

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

期刊

BIOTECHNOLOGY AND BIOENGINEERING
卷 110, 期 8, 页码 2073-2085

出版社

WILEY-BLACKWELL
DOI: 10.1002/bit.24903

关键词

cell-free protein synthesis; flagellin; virus-like particle; vaccine adjuvant; VLP vaccines; enhanced TLR stimulation

资金

  1. NIH Stanford U19 consortium [AI057229, AI090019]

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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 mu g/mL and the product had much higher affinity for the TLR5 receptor (EC50=2.4 +/- 1.4pM) 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. (c) 2013 Wiley Periodicals, Inc.

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