Abstract
Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.
Highlights
The heterologous expression and purification of proteins allows their biochemical characterization, use in industrial processes, and development of commercial goods (Rosano and Ceccarelli, 2014)
Using an expression vector conferring an N-terminal Strep-Tag to the cytoplasmic NqrA subunit, production of V. cholerae NADH:quinone oxidoreductase (NQR) produced in V. natriegens was confirmed with western blotting followed by immune detection (Figure 1A)
Strep-tagged NqrA with an apparent molecular mass of 53.5 kDa was detected in membranes from V. natriegens cells transformed with pNqrST and with purified NQR-ST complex, but not in membranes from V. natriegens cells transformed with the control vector, pBAD-TOPO
Summary
The heterologous expression and purification of proteins allows their biochemical characterization, use in industrial processes, and development of commercial goods (Rosano and Ceccarelli, 2014). The traditional microbial system for heterologous expression of proteins is Escherichia coli with high levels of gene expression and scalability of experiments. The production of recombinant proteins, especially membrane-bound proteins, represents the bottleneck of many biotechnological processes. We introduce Vibrio natriegens as a new host for heterologous expression of multisubunit membrane proteins in their active state.
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