Application of an E. coli signal sequence as a versatile inclusion body tag

Microbial Cell Factories

Research

Diane Houben¹  H. Bart van den Berg van Saparoea¹  Wouter S. P. Jong²  Joen Luirink²  Jan-Willem de Gier³  David Vikström⁴ 

[1] Abera Bioscience AB, 11145, Stockholm, Sweden;Abera Bioscience AB, 11145, Stockholm, Sweden;Department of Molecular Cell Biology, Section Molecular Microbiology, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands;Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691, Stockholm, Sweden;Xbrane Biopharma AB, 11145, Stockholm, Sweden;

关键词: Inclusion bodies;    Fusion tag;    Insolubility;    Aggregation;    Heterologous protein production;    E. coli;    Signal peptide;    Twin-arginine translocation pathway;   

DOI  :  10.1186/s12934-017-0662-4

 received in 2016-05-31, accepted in 2017-03-10,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundHeterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form.ResultsWhen screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins.ConclusionsWe present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

【 授权许可】

CC BY   
© The Author(s) 2017