Construction of a Strain for Promoting Production of Small Molecule Antibodies in Periplasmic Space of <i>Escherichia coli</i>

doi:10.13523/j.cb.2001001

China Biotechnology

2020, Vol. 40

Issue (5): 48-56 DOI: 10.13523/j.cb.2001001

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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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

Received: 02 January 2020 Published: 02 June 2020

ZTFLH:

Q815

Corresponding Authors: Yong-qing CHENG E-mail: ycheng@triprime.com

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	Mei TONG
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Cite this article:

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.

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

Fig.1 Expression levels of Fab' were related to vectors selection in BL21(DE3) (a) Representive Western blots of periplasmic Fab' expression levels when constructed in different vectors (b) Comparison of average signal intensities of assembled Fab' according to Western blot analysis (c)A schematic diagram of the plasmid construction of Fab' in pET-30a Data were expressed as mean± S.E.M. and collected from at least three colonies. *P<0.05 compared with all other strains; 1:Un-induced;2:Induced

Fig.2 Expression levels of Fab' were relatedto signal peptide and different VH-VL constructions in BL21(DE3) (a) Western blot of different constructs OmpA-VH-OmpA-VL: The variable region of heavy chain was put before the variable region of light chain; OmpA-VL-OmpA-VH: The variable region of light chain was put before the variable region of heavy chain;MalE-VL-OmpA-VH: The signal peptide was changed to MalE before the heavy chain; (b) A schematic diagram of switched construction of Fab' in pET-30a;1: Induced in 30℃ for 16h;2: Induced in 37℃ for 16h

Fig.3 Expression levels of Fab' were related to co-expression with other molecular chaperones in BL21(DE3) Nuclease: Nuclease was coexpressed wih Fab'; Dsbc: Chaperone protein Dsbc was coexpressed wih Fab'; PPIB: Chaperone protein PPIB was coexpressed wih Fab'; Skp: Chaperone protein Skp was coexpressed wih Fab'; hPDI: Chaperone protein hPDI was coexpressed wih Fab'; 1: Induced in 30℃ for 16h; 2: Induced in 37℃ for 16h

Fig.4 Periplasmic extraction was purified by Protein L chromatography (a) An elution peak of protein L was recorded (b) Sample, flow-through and elute of protein L were analyzed by SDS-PAGE 1:Sample; 2:Flow-through; 3:Elute

Fig.5 The affinity and neutralization activity of Fab' to human TNF-α (a) The affinity of Fab' to human TNF-α antigen was tested by ELISA (b) The neutralization activity of Fab' in vitro was tested on L929 cells


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