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Applied Microbiology and Biotechnology

, Volume 103, Issue 8, pp 3341–3353 | Cite as

A general platform for efficient extracellular expression and purification of Fab from Escherichia coli

  • Manyu Luo
  • Meiqi Zhao
  • Cedric Cagliero
  • Hua Jiang
  • Yueqing Xie
  • Jianwei Zhu
  • Hui Yang
  • Mengxiao Zhang
  • Ying Zheng
  • Yunsheng Yuan
  • Zixiu Du
  • Huili LuEmail author
Biotechnological products and process engineering

Abstract

Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.

Keywords

Fab phoA STII Extracellular production E. coli 

Notes

Funding information

This work was supported in part by the National Natural Science Foundation of China (No. 81773621 to Zhu J.) and the Science and Technology Commission of Shanghai Municipality (No. 17431904500 & 17ZR1413700 to Lu H.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animal experiments.

Supplementary material

253_2019_9745_MOESM1_ESM.pdf (310 kb)
ESM 1 (PDF 309 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of PharmacyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Jecho Laboratories, Inc.FrederickUSA
  3. 3.School of PharmacyShanghai Jiao Tong UniversityShanghaiChina

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