Heterologous gene expression in a membrane-protein-specific system

We have constructed an expression system for heterologous proteins which us es the molecular machinery responsible for the high level production of bac teriorhodopsin in Halobacterium salinarum, Cloning vectors were assembled t hat fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that per mitted transfer of fused genes into H, salinarum expression vectors. Gene f usions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxy l-terminal fusion with the catalytic subunit of the Escherichia coli aspart ate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast cu mating fact or receptor, Ste2, Characterization of the expression of these fusions reve aled that the bop gene coding region contains previously undescribed molecu lar determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-term inal coding region significantly decreased bop gene mRNA and protein accumu lation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E, coli codon u sage bias did not limit the system's potential for high level expression. T he work presented describes initial efforts in the development of a novel h eterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins. ( C) 1999 Academic Press.