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.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
小玉完成签到,获得积分20
刚刚
ket完成签到,获得积分10
1秒前
超帅的心锁完成签到,获得积分20
1秒前
1秒前
1秒前
1秒前
ygng_uua发布了新的文献求助10
1秒前
177ycd发布了新的文献求助10
1秒前
呱呱发布了新的文献求助10
1秒前
流云发布了新的文献求助10
1秒前
unite 小丘发布了新的文献求助10
2秒前
文献啊文献完成签到,获得积分10
2秒前
Cc发布了新的文献求助10
3秒前
炙热尔烟完成签到,获得积分10
3秒前
3秒前
3237507683完成签到,获得积分20
4秒前
4秒前
卜婉君发布了新的文献求助10
4秒前
5秒前
5秒前
VAN喵发布了新的文献求助10
5秒前
5秒前
5秒前
今天也在痛苦上学完成签到,获得积分10
6秒前
Hello应助面包康采纳,获得10
6秒前
6秒前
充电宝应助为学日益采纳,获得10
6秒前
02发布了新的文献求助10
6秒前
花陌发布了新的文献求助10
6秒前
7秒前
7秒前
7秒前
俭朴新瑶完成签到,获得积分10
8秒前
8秒前
8秒前
标致的坤完成签到,获得积分10
8秒前
柚子蟹完成签到,获得积分10
8秒前
8秒前
9秒前
whh发布了新的文献求助10
9秒前
高分求助中
Picture Books with Same-sex Parented Families: Unintentional Censorship 700
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
Nucleophilic substitution in azasydnone-modified dinitroanisoles 500
不知道标题是什么 500
Indomethacinのヒトにおける経皮吸収 400
Phylogenetic study of the order Polydesmida (Myriapoda: Diplopoda) 370
Effective Learning and Mental Wellbeing 300
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3974643
求助须知:如何正确求助?哪些是违规求助? 3519094
关于积分的说明 11196979
捐赠科研通 3255182
什么是DOI,文献DOI怎么找? 1797700
邀请新用户注册赠送积分活动 877100
科研通“疑难数据库(出版商)”最低求助积分说明 806130