CONSTRUCTION OF AN EFFICIENT BACILLUS-SUBTILIS SYSTEM FOR EXTRACELLULAR PRODUCTION OF HETEROLOGOUS PROTEINS

An efficient expression/secretion vector, designated pM2Veg, was const ructed for extracellular production of heterologous proteins in Bacill us subtilis. To construct pM2Veg, a synthetic Cassette, the Veg casset te carrying: (1) the strong vegetative vegI promoter from B. subtilis, (2) the Escherichia coli lac operator, (3) the B. subtilis consensus ribosome-binding site, (4) the Staphylococcal protein A leader sequenc e, (5) a cloning region for insertion of foreign genes, (6) translatio nal stop codons in all three reading frames, and (7) the gnt transcrip tional 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 il lustrated using two widely different proteins: the endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi and human epidermal gro wth factor (hEGF). Levels of Eng and hEGF measured in culture supernat ant samples of B. subtilis transformants harboring recombinant constru cts formed between pM2Veg and the cenA and hEGF genes were 8.3 U ml(-1 ) and 7.0 mg 1(-1), respectively. The Eng activity is more than four t imes higher than the yield from the best cenA recombinant construct pr eviously reported, and the hEGF data represents the first successful e xpression of the factor in B. subtilis. (C) 1998 Elsevier Science B.V. All rights reserved.