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.

Use of recombinant flagellin in oil-in-water emulsions enhances hemagglutinin-specific mucosal IgA production and IL-17 secreting T cells against H5N1 avian influenza virus infection

“…In the present study we found that recombinant FliC protein can serve as a mucosal adjuvant for H5N1 subunit HA vaccine development. We observed that recombinant FliC protein expressed in E. coli was mostly soluble, yet contained a full-length form (51 kDa) and a truncated form (47 kDa) similar to that recently reported for a cell-free protein synthesis system [27]. Bacterial flagellin contains four domains (D0, D1, D2, D3), with the D0 domain congregating in a compact and stable structure inside the flagellum [28].…”

Functional TLR5 Genetic Variants Affect Human Colorectal Cancer Survival

“…Because high IL-6 levels in serum and tumor tissue link with an unfavorable colorectal cancer prognosis due to a tumor-promoting role of IL-6 via STAT3 (28), it is tempting to speculate that rs5744174/F616L may directly favor the observed better colorectal cancer survival (cf. Mechanistic studies into the molecular basis for the observed TLR5 genotype-dependent differences in responsiveness to the prototypical Salmonella flagellin, including the role of the D 0 domain (29), as well as the molecular basis for the sensitivity of TLR5 to interspecies flagellin variation (cf. Fig.…”

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery

“…Recently, an Escherichia coli-based cell-free protein synthesis (CFPS) system was successfully developed for the production and modification of VLPs (11,12). CFPS enables rapid production of VLPs at high yields (∼0.5 g/L) in a few hours.…”

“…Flagellin and GMCSF were used as example proteins for conjugation. CFPS provides a facile means for site-specific introduction of nnAAs with an alkyne moiety into flagellin (HPG, homopropargylglycine) (11) and GMCSF (PPF, p-propargyloxy-phenylalanine) (14), as well as a nnAA with an azide moiety (AHA, azidohomoalanine) into the L76 site near the tip of the spike region on the VLP surface. This approach enabled the direct coupling of flagellin and GMCSF to the VLPs by using Cu(I)-catalyzed [3+2] cycloaddition click chemistry (SI Appendix, Fig.…”