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

Abstract

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.

Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior

“…The pAIDA1 vector22 (Fig. 1a), including the wild type signal peptide and β-barrel (AIDA c ), was engineered to facilitate the identification of correct surface display by introducing two reporter sequences (His 6 and Myc tags) flanking the passenger (Fig.…”

“…The plasmid pAIDA122 was used for the surface expression of tyrosinases. This plasmid uses the AIDA-I surface expression system fused to a recombinant passenger protein flanked by an N-terminal His 6 detection tag and a C-terminal Myc detection tag (Fig.…”

“…The next day, the membrane was incubated for 60 min in PBST containing 10 g/L bovine serum albumin and 0.5 μg/mL antibody (THE TM c-Myc [HRP], GenScript). After washing in PBST, the membranes were developed as previously described1722.…”

Surface Expression of ω-Transaminase in Escherichia coli

“…Plasmid pET28aϩ containing the gene for an Arthrobacter citreus -transaminase variant (AcTA) (17) was used for intracellular production of transaminase, as well as the template for amplification of the transaminase gene. The pAIDA1 plasmid (18) was used for surface expression of the transaminase. This plasmid carries genes encoding the signal peptide and ␤-barrel (AIDA c ) of AIDA-I, with a cloning site for a recombinant passenger protein between the genes.…”

“…The host organism for surface expression was an OmpT-negative strain derived from E. coli K-12 0:17 (20) (0:17⌬OmpT), which has previously been found suitable for recombinant surface expression using the AIDA autotransporter (13,18,21). E. coli strain BL21(DE3) was used for production of purified transaminase using the T7-based promoter of pET28aϩ.…”