Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum

Abstract

Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.The display of heterologous proteins or peptides on the surface of prokaryotic or eukaryotic cells, especially bacterial and yeast cells (8), enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications, including live vaccine development, antibody production, peptide library screening, the use of environmental bioadsorbents, and whole-cell biocatalysis (12). A variety of surface anchoring motifs have been used to establish display systems, including various outer membrane proteins, lipoproteins, autotransporters, subunits of surface appendages, and S-layer proteins (12, 16). The efficiency of surface display systems are highly related to the characteristics of the carrier protein, passenger protein, host cell, and fusion method (17).Lpp-OmpA is an efficient surface display system developed by Georgiou et al. (9), which has been used successfully to anchor a variety of proteins, including some enzymes, onto the cell surface (10, 21). This display system allows C-terminal fusion of the passenger proteins and consists of two key anchoring motifs: (i) the signal sequence and the first nine amino acids of E. coli lipoprotein (Lpp), to target the proteins to the inner face of the outer membrane, and (ii) the transmembrane region (amino acids 46 to 159) of E. coli outer membrane protein A (OmpA), to conduct the proteins across the outer membrane. Since the Lpp-OmpA mode combines the anchoring capabilities of lipoprotein and outer membrane protein, it has outstanding advantages in high surface display efficiency and st...

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