Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination. Issue 10 (4th March 2016)

• Record Type: Journal Article
• Authors: Tamaki, Yukihiro; Harakuni, Tetsuya; Yamaguchi, Rui; Miyata, Takeshi; Arakawa, Takeshi
• Is Part Of: Vaccine. Volume 34:Issue 10(2016)
• Page Start: 1268
• Page End: 1274
• Publication Date: 2016-03-04
• Subjects: COMP cartilage oligomeric matrix protein -- CT cholera toxin -- CTA cholera toxin A subunit -- CTB cholera toxin B subunit -- GdnHCl guanidine hydrochloride -- GM1 monosialoganglioside GM1 -- HMM high-molecular-mass -- IBs inclusion bodies -- RP refolding procedure; Cholera toxin B subunit -- Pentamer -- Multisubunit protein refolding -- Dialysis; Vaccines -- Periodicals; 615.372
• DOI: 10.1016/j.vaccine.2016.01.034
• Languages: English
• ISSNs: 0264-410X

Abstract

Highlights: Cholera toxin B subunit was refolded from Escherichia coli inclusion bodies. The refolded protein was predominantly soluble high-molecular-mass aggregates. Contaminant removal efficiently regenerated homogeneous pentamer. Regenerated pentamer was functionally indistinguishable from choleragenoid. Abstract: The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli . Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5 mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5 mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae . This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.