Increased expression of Brevibacterium sterolicum cholesterol oxidase in 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 codons. These changes resulted in a 60-fold improvement of expression level. N-terminal sequencing revealed that the E. coli produced cholesterol oxidase signal peptide is cleaved 6 amino acids closer to the N-terminus than in B. sterolicum. The recombinant E. coli produced protein is composed of 513 amino acids with a calculated Mr of 55,374. The kinetic rate constants of the recombinant protein and the B. sterolicum produced cholesterol oxidase are identical.