A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

Microbial Cell Factories

Research

Thi-Huyen Do¹  Martin Gustavsson²  Johan Jarmander²  Gen Larsson²  Patrik Samuelson² 

[1] Institute of Biotechnology, Vietnamese Academy of Science and Technology, VAST, 18-Hoang Quoc Viet, Hanoi, Cau Giay, Vietnam;School of Biotechnology, Division of Bioprocess Technology, Royal Institute of Technology,KTH, 106 91, Stockholm, SE, Sweden;

关键词: AIDA;    Surface expression;    Autotransport;    Escherichia coli;    Proteolysis;    Detection tag;   

DOI  :  10.1186/1475-2859-11-118

 received in 2012-06-06, accepted in 2012-08-18,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express.ResultsThe surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium.ConclusionsExpression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

【 授权许可】

Unknown   
© Jarmander et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.