INCREASED EXPRESSION OF BREVIBACTERIUM-STEROLICUM CHOLESTEROL OXIDASEIN ESCHERICHIA-COLI BY GENETIC-MODIFICATION

To improve expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli, we utilized the T7lac promoter and modified the gene to encode the first 21 amino acids with high-expression E. coli c odons. These changes resulted in a 60-fold improvement of expression l evel. N-terminal sequencing revealed that the E. coli produced cholest erol oxidase signal peptide is cleaved 6 amino acids closer to the N-t erminus than in B. sterolicum. The recombinant E. coli produced protei n is composed of 513 amino acids with a calculated M-r of 55,374, The kinetic rate constants of the recombinant protein and the B. sterolicu m produced cholesterol oxidase are identical, (C) 1998 Academic Press.