Cloning and Expression of Brevibacterium sp. DGCDC-82 Cholesterol Oxidase Gene in Escherichia coli / 微生物学通报

ABSTRACT

Based on DNA sequence encoding cholesterol oxidase reported on the NCBI,cholesterol oxidase gene was cloned from Brevibacterium sp. DGCDC-82 by PCR methods,which showed homology of 98% to the previously reported cholesterol oxidase gene from Brevibacterium sterolicum ATCC21387. Subsequently,the resulting products were digested with NcoI and EcoRI and ligated to the pET28a vector by T4 DNA ligase.The recombinant plasmid,pET28a-choB,was transformed into Escheriehia coli BL21-CodonPlus(DE3)-RP which contain extra copies of the argU and proL genes.The positive clone was induced with IPTG,and enzyme expressed in BL21-CodonPlus(DE3)-RP,the enzyme activity was about 340U/L.The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 55kD protein was obtained,which accounted for about 16% of the total cell protein.