| An efficient expression/secretion vector, designated pM2Veg, was constructed for extracellular production of heterologous proteins in Bacillus subtilis. To construct pM2Veg, a synthetic cassette, the Veg cassette carrying: (i) the strong vegetative veg I promoter ("Site I" of the veg promoter) from B. subtilis, (ii) the Escherichia coli lac operator, (iii) the B. subtilis consensus ribosome-binding site, (iv) the Staphylococcal protein A leader sequence, (v) a cloning region for insertion of foreign genes, (vi) translational stop codons in all three reading frames, and (vii) the gnt transcriptional terminator, was cloned into a derivative of the stable pRB373 B. subtilis /E. coli shuttle plasmid, the pM2 vector. The application of pM2Veg to effect secretory production of heterologous proteins was illustrated using two widely different proteins: the endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi and human epidermal growth factor (hEGF). Levels of Eng and hEGF measured in culture supernatant samples of B. subtilis transformants harboring recombinant constructs formed between pM2Veg and the cenA and hEGF genes were 8.3 U ml-1 and 7.0 mg l-1, respectively. The Eng activity is more than 4 times higher than the yield from the best cenA recombinant construct previously reported, and the hEGF data represents the first successful expression of the factor in B. subtilis. The success of employing the novel pM2Veg vector in expressing both Eng and hEGF, which are widely different proteins with regard to their sizes and origins, suggests that the vector may be employed to express a wide variety of heterologous proteins in B. subtilis .;To enable pM2Veg and its derivatives to be transformed into B. subtilis strains, a spontaneous point mutation, involving a conversion of an A (underlined) to a G, located in a palindromic sequence, 5' TTGTACAA 3', which was identified at the -10 region of the veg I promoter, was found to be essential. The observation suggested that the native veg I promoter relied on some modification for protection, whereas a recombinant veg I promoter, which lacked such a modification, depended upon the aforementioned spontaneous point mutation for its maintenance in B. subtilis. Elucidation of the cause and manifestation of the spontaneous mutation may lead to a better understanding of the relationship between gene expression and regulation, and restriction and modification in B. subtilis. |
