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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (5): 48-56    DOI: 10.13523/j.cb.2001001
技术与方法     
促进大肠杆菌周质空间小分子抗体表达的菌种构建方法*
童梅,程永庆(),刘金毅,徐晨
北京三元基因药业股份有限公司 北京市长效干扰素工程技术研究中心 北京 102600
Construction of a Strain for Promoting Production of Small Molecule Antibodies in Periplasmic Space of Escherichia coli
TONG Mei,CHENG Yong-qing(),LIU Jin-yi,XU Chen
Beijing Tri-Prime Gene Pharmaceutical Co., Ltd. Beijing Engineering Research Center of Long-acting Interferon, Beijing 102600,China
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摘要：

目的:构建一株表达TNF-α Fab'抗体的大肠杆菌工程菌,并设计一种高效实用的策略以促进大肠杆菌周质空间的可溶性Fab'抗体表达。方法:首先,通过更换不同表达载体,改变轻链和重链顺序,更换信号肽,共表达分子伴侣(Skp)、二硫键合成酶(Dsbc)、肽基辅氨酰顺反异构酶(PPIB)、二硫键异构酶(hPDI)、核酸酶(Nuclease),以评估对Fab'抗体表达量的改善。其次,纯化表达的Fab'抗体。通过周质提取、Q阴离子交换柱净化、苯基柱捕获、Protein L柱亲和三步纯化方案得到高纯度的Fab'抗体。最终将纯化后的Fab'抗体进行亲和力测定。结果:提高正确组装的Fab'抗体表达量的策略有——将目的蛋白构建至pET-30a载体;重链在前、轻链在后;轻、重链采用相异的信号肽;共表达hPDI。周质提取液中的Fab'抗体浓度达到588.0mg/L提取液,纯化后产量可达28.2mg/L发酵液,总回收率为32.0%,纯度为90.9%。Fab'抗体亲和力为(5.8±3.0)×10-9mol/L,体外细胞学活性IC50为(5.2±2.4)×10-11mol/L。结论:通过大肠杆菌工程菌分子构建方式的优化,得到了一株高效表达可溶性Fab'抗体的工程菌株,为可溶性小分子抗体的规模化生产奠定了研究基础。

关键词： Fab;  TNF-α;  抗肿瘤坏死因子;  大肠杆菌表达;  周质空间表达;  原核生产体系    
Abstract:

Objective: To construct an E. coli strain engineered for expressing TNF-α Fab' antibody, and to design an efficient and practical strategy to promote the expression of soluble Fab' antibody in the periplasmic space. Methods: First, the strategies were selected as changing different expression vectors, changing the order of light and heavy chains, changing signal peptides, co-expressing molecular chaperones (Skp), disulfide bond synthetase (Dsbc), peptidyl coenzyme cis-trans isomerase (PPIB), disulfide isomerase (hPDI), and nuclease to evaluate the improvement of Fab' antibody expression. Second, the expressed Fab' antibody was purified. High-purity Fab' antibodies were obtained through a three-step purification of periplasmic extraction, including Q anion exchange column purification, phenyl column capture, and Protein L column affinity purification. Finally, the affinity of the purified Fab' antibody was determined. Results: The best strategy to increase the expression of correctly assembled Fab' antibodies suggested the target protein was constructed into pET-30a vector; the heavy chain was in the front and the light chain was in the rear; light and heavy chains used different signal peptides, and co-expression of hPDI. The Fab' antibody concentration in the periplasmic extract reached 588.0mg/L, and the yield after purification was up to 28.2mg/L in the fermentation broth. The total recovery rate was 32.0% and the purity of Fab' was 90.9%. The affinity of Fab' antibody was (5.8±3.0)×10 -9mol/L, and in vitro activity IC50 was (5.2±2.4)×10 -11mol/L. Conclusion: Through the optimization of the molecular construction, an engineered E. coli strain with high level of soluble Fab' antibody expression was obtained.

Key words: Fab    TNF-α Anti-tumor necrosis factor    E.coli expression    Periplasmic space expression    Prokaryotic production system
收稿日期: 2020-01-02 出版日期: 2020-06-02
ZTFLH:  Q815  
基金资助: * 北京市科技计划(Z141100000514008)
通讯作者: 程永庆     E-mail: ycheng@triprime.com
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引用本文:

童梅,程永庆,刘金毅,徐晨. 促进大肠杆菌周质空间小分子抗体表达的菌种构建方法*[J]. 中国生物工程杂志, 2020, 40(5): 48-56.

TONG Mei,CHENG Yong-qing,LIU Jin-yi,XU Chen. Construction of a Strain for Promoting Production of Small Molecule Antibodies in Periplasmic Space of Escherichia coli. China Biotechnology, 2020, 40(5): 48-56.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001001    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I5/48

图1  不同的载体选择对Fab'在BL21(DE3)周质中表达水平的影响
图2  改变重链位置及信号肽可影响Fab'在BL21(DE3)中的表达水平
图3  与不同分子伴侣共表达可提高BL21(DE3)的Fab'表达量
图4  周质提取物经过Protein L柱进行亲和层析
图5  Fab'抗体与人TNF-α抗原的亲和力及体外中和活性检测
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