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. 2012 Sep 3:11:118.
doi: 10.1186/1475-2859-11-118.

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

Johan Jarmander  1 , Martin Gustavsson, Thi-Huyen Do, Patrik Samuelson, Gen Larsson
Affiliations

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

Johan Jarmander et al. Microb Cell Fact. .

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.

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Figures

Figure 1
Figure 1
Schematic representations of pAIDA1 and the surface expressed fusion proteins of pAIDA1 and pDT1. Left: The surface expression vector pAIDA1 based on the AIDA-I autotransporter. Expression is under the control of the lacUV5 promoter, and two detection tags (His6 and Myc) with adjacent protease cleavage recognition sites (for 3C and TEV) flank the passenger protein. Top right: Matured fusion protein at the cell surface from vector pAIDA1. Bottom right: Matured fusion protein at the cell surface from vector pDT1.
Figure 2
Figure 2
Flow cytometry histograms of expressed SefA on the surface of E. coli. Left: Detection of His6 tag by the THETM His Tag [FITC] antibody. Negative control (red) consisting of cells lacking vector showed a mean fluorescence of 0.6. Cells harboring pDT1-SefA (green) showed a mean fluorescence of 10.5 in the positive population. Cells harboring pAIDA1-SefA (blue) show a mean fluorescence of 15.3. Right: Detection of Myc tag by Anti-c-Myc [SureLight® Allophycocyanin] antibody. Negative control (red) showed a mean fluorescence of 0.6 and cells harboring pAIDA1-SefA (blue) show a mean fluorescence of 15.4.
Figure 3
Figure 3
Flow cytometry histograms of expressed H:gm on the surface of E. coli. Left: Detection of His6-tag by the THETM His Tag [FITC] antibody. Negative control (red) consisting of cells lacking vector showed a mean fluorescence of 0.6. Cells harboring pDT1-H:gm (green) showed a mean fluorescence of 1.4. Cells harboring pAIDA1-H:gm (blue) show a mean fluorescence of 1.5. Right: Detection of Myc-tag by Anti-c-Myc [SureLight® Allophycocyanin] antibody. Negative control (red) showed a mean fluorescence of 0.6 and cells harboring pAIDA1-H:gm (blue) show a mean fluorescence of 7.6.
Figure 4
Figure 4
Western blot showing distribution of SefA and H:gm from pDT1 in cellular fractions. Detection through an anti-His6 antibody conjugated with horseradish peroxidase. Lanes 1 and 6: Size marker. Lanes 2 and 7: Outer membrane fraction of pAIDA1 without passenger (positive control). Lane 3: pDT1-SefA soluble fraction. Lane 4: pDT1-SefA inner membrane fraction. Lane 5: pDT1-SefA outer membrane fraction. Lane 8: pDT1-H:gm soluble fraction. Lane 9: pDT1-H:gm inner membrane fraction. Lane 10: pDT1-H:gm outer membrane fraction.
Figure 5
Figure 5
Western blot showing distribution of SefA and H:gm from pAIDA1 in cellular fractions. Left: Detection through an anti-His6 antibody conjugated with horseradish peroxidase. Lanes 1 and 6: Size marker. Lanes 2 and 7: Outer membrane fraction of pAIDA1 without passenger (positive control). Lane 3: pAIDA1-SefA soluble fraction. Lane 4: pAIDA1-SefA inner membrane fraction. Lane 5: pAIDA1-SefA outer membrane fraction. Lane 8: pAIDA1-H:gm soluble fraction. Lane 9: pAIDA1-H:gm inner membrane fraction. Lane 10: pAIDA1-H:gm outer membrane fraction. Right: Detection through an anti-Myc antibody conjugated with horseradish peroxidase. Lanes 1 and 6: Size marker. Lane 2: pAIDA1-SefA soluble fraction. Lane 3: pAIDA1-SefA inner membrane fraction. Lane 4: pAIDA1-SefA outer membrane fraction. Lane 5 and 10: Outer membrane fraction of pAIDA1 without passenger (positive control). Lane 7: pAIDA1-H:gm soluble fraction. Lane 8: pAIDA1-H:gm inner membrane fraction. Lane 9: pAIDA1-H:gm outer membrane fraction.
Figure 6
Figure 6
Tertiary structure of H:gm. Atomic model of the Salmonella enterica flagellar protein H:gm by electron cryomicroscopy [PDB: 1UCU] drawn in PyMOL. The protein consists of 494 aa and forms a tubular tertiary structure with the N- and C-terminals positioned close to each other.
See this image and copyright information in PMC

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