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以大肠杆菌分批补料培养方式实现每升克级表达人类分子伴侣域 Bri2 BRICHOS。

Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture.

机构信息

Department of Biosciences and Nutrition, Karolinska Institutet, Neo, 141 86, Huddinge, Sweden.

Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.

出版信息

Microb Cell Fact. 2021 Jul 30;20(1):150. doi: 10.1186/s12934-021-01638-8.

DOI:10.1186/s12934-021-01638-8
PMID:34330289
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8325310/
Abstract

BACKGROUND

The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli.

RESULTS

A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures.

CONCLUSIONS

The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.

摘要

背景

人类 Bri2 BRICHOS 结构域抑制淀粉样形成和毒性，可作为治疗淀粉样疾病的药物。为了将其转化为临床应用，需要大量正确折叠的重组人（rh）Bri2 BRICHOS。为了提高 rh Bri2 BRICHOS 的表达和可溶性水平，将其与 NT融合，NT是一种来源于蜘蛛丝蛋白 N 端结构域的可溶性标签，可显著提高目标蛋白的表达水平和可溶性。为了进一步提高表达水平并达到 g/L 范围，这是在工业规模上进行经济生产的前提条件，我们为大肠杆菌开发了一种分批补料表达方案。

结果

建立并系统优化了 NT*-Bri2 BRICHOS 的分批补料生产方法。这种逐步改进导致表达水平高达 18.8 g/L。表达后，通过色谱方法对 NT*-Bri2 BRICHOS 进行纯化，最终收率高达 6.5 g/L。去除 NT*-标签并分离成不同的寡聚体后，活性测定验证了分批补料生产的不同组装状态的 rh Bri2 BRICHOS 具有与从摇瓶培养物中分离出的参考蛋白相同的抑制纤维状和非纤维状蛋白聚集的能力。

结论

本工作中开发的方案允许使用可溶性增强 NT*-标签作为融合伴侣大量生产 rh Bri2 BRICHOS，这是进行临床前研究所必需的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/209d4662d141/12934_2021_1638_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/105905700dd1/12934_2021_1638_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/d9e26fea0777/12934_2021_1638_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/69db7a389011/12934_2021_1638_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/875c967e0d5b/12934_2021_1638_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/80ec105af35a/12934_2021_1638_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/60080bcc03f4/12934_2021_1638_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/209d4662d141/12934_2021_1638_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/105905700dd1/12934_2021_1638_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/d9e26fea0777/12934_2021_1638_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/69db7a389011/12934_2021_1638_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/875c967e0d5b/12934_2021_1638_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/80ec105af35a/12934_2021_1638_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/60080bcc03f4/12934_2021_1638_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b022/8325310/209d4662d141/12934_2021_1638_Fig7_HTML.jpg

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BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells.
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