title: Increased expression of Brevibacterium sterolicum cholesterol oxidase in Escherichia coli by genetic modification.
abstract: 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.
authors.affiliation: Department of Chemistry, State University of New York, Stony Brook, New York 11794-3400, USA. nicole.sampson@sunysb.edu
authors.forename: N S
authors.initials: NS
authors.lastname: Sampson
authors.forename: X
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics; D002789:Cholesterol Oxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002789:Cholesterol Oxidase