Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.
Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.
复制标题
由大肠杆菌包涵体重组而成的霍乱毒素 B 亚基五聚体，用于疫苗接种。
DOI：
10.1016/j.vaccine.2016.01.034
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发表时间：
2016
期刊：
影响因子：
5.5
通讯作者：
Arakawa T
中科院分区：
文献类型：
--
作者：
Tamaki Y;Harakuni T;Yamaguchi R;Miyata T;Arakawa T
霍乱毒素B亚基（CTB）以其五聚体形式从大肠杆菌分泌，如果其前导肽被一个e取代。coliorigin。然而，当分子发生突变或与外源肽融合时，五聚体的分泌通常会严重受损。因此，我们尝试从e的包涵体（ib）再生五聚体CTB。杆菌。逐步透析溶解在盐酸胍中的IBs主要产生可溶性高分子质量（HMM）聚集体和只有一小部分五聚体。评估了三种重组均相五聚体分子的方法：(i)使用五聚体线圈-线圈融合伙伴，期望其作为组装核心；（ii）优化重折叠过程中的蛋白质浓度；（三）在再折叠前去除污染物。线圈融合有一定的效果，但仍产生了大量的HMM聚集体。从0.05 mg/mL到5 mg/mL的蛋白质浓度变化几乎没有影响。相比之下，在再折叠之前消除污染物具有强大的效果，并且只有五聚体再生，没有可检测到的HMM聚集体。令人惊讶的是，当污染物被去除时，蛋白质在再折叠时的浓度高达5 mg/mL，对再折叠没有不利影响。再生的五聚体在生化和免疫学特性上与me分泌的CTB没有区别。来自霍乱弧菌的彩色类霍乱杆菌。本研究提供了一种简单而高效的CTB五聚体聚合策略，具有高度均匀的分子构象，为设计CTB衍生物和CTB融合抗原提供了可能。
The cholera toxin B subunit (CTB) is secreted in its pentameric form fromEscherichia coliif its leader peptide is replaced with one ofE. coliorigin. 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) ofE. 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 fromE. colior choleragenoid fromVibrio 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.