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Escherichia coli ‐based cell free production of flagellin and ordered flagellin display on virus‐like particles
Author(s) -
Lu Yuan,
Welsh John P.,
Chan Wei,
Swartz James R.
Publication year - 2013
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24903
Subject(s) - flagellin , tlr5 , escherichia coli , biology , biochemistry , chemistry , microbiology and biotechnology , receptor , innate immune system , toll like receptor , gene
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.