Email updates

Keep up to date with the latest news and content from Microbial Cell Factories and BioMed Central.

Open Access Research

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

Johan Jarmander1, Martin Gustavsson1, Thi-Huyen Do2, Patrik Samuelson1 and Gen Larsson1*

Author Affiliations

1 School of Biotechnology, Division of Bioprocess Technology, Royal Institute of Technology, KTH, Stockholm, SE, 106 91, Sweden

2 Institute of Biotechnology, Vietnamese Academy of Science and Technology, VAST, 18-Hoang Quoc Viet, Hanoi, Cau Giay, Vietnam

For all author emails, please log on.

Microbial Cell Factories 2012, 11:118  doi:10.1186/1475-2859-11-118

Published: 3 September 2012

Abstract

Background

The 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.

Results

The 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.

Conclusions

Expression 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.

Keywords:
AIDA; Surface expression; Autotransport; Escherichia coli; Proteolysis; Detection tag