A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli

Microbial Cell Factories

Research

Yulu Wu¹  Cheng Cheng¹  Lupeng Cui¹  Bingfang He²  Tianyue Jiang³  Shanshan Wu⁴ 

[1] School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, 211816, Nanjing, People’s Republic of China;School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, 211816, Nanjing, People’s Republic of China;School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, 211816, Nanjing, People’s Republic of China;School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, 211816, Nanjing, People’s Republic of China;Wuxi AppTec (Suzhou) Testing Technology Co.,Ltd., 1336 Wuzhong Avenue, Xinzhiyuan Building B, Wuzhong District, 215104, Suzhou, Jiangsu, China;

关键词: Secretory expression;    Fusion tag;    β;    Heterologous proteins;    Escherichia coli;   

DOI  :  10.1186/s12934-017-0845-z

 received in 2017-08-23, accepted in 2017-12-13,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner.MethodsBased on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags.ResultsThe expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase.ConclusionsObservations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins.

【 授权许可】

CC BY   
© The Author(s) 2017