title: Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles.
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.
authors.affiliation: Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, California 94305-5025, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lu
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
authors.forename: Wei
authors.initials: W
authors.lastname: Chan
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000276:Adjuvants, Immunologic / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005408:Flagellin / Q000302:isolation & purification* / Q000378:metabolism; D006512:Hepatitis B Core Antigens / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D012486:Salmonella typhimurium / Q000235:genetics; D051198:Toll-Like Receptor 5 / Q000819:agonists*; D058425:Vaccines, Virus-Like Particle / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D006512:Hepatitis B Core Antigens; D051198:Toll-Like Receptor 5; D058425:Vaccines, Virus-Like Particle; D005408:Flagellin