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Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination

五聚体 大肠杆菌 霍乱毒素 霍乱弧菌 包涵体 化学 蛋白质亚单位 微生物学 融合蛋白 生物 生物化学 生物物理学 细菌 重组DNA 遗传学 基因
作者
Yukihiro Tamaki,Tetsuya Harakuni,Rui Yamaguchi,Takeshi Miyata,Takeshi Arakawa
出处
期刊:Vaccine [Elsevier BV]
卷期号:34 (10): 1268-1274 被引量:8
标识
DOI:10.1016/j.vaccine.2016.01.034
摘要

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.
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