Cloning, Expression and Purification of Creatinase.

BACKGROUND

ATCC12633 can produce creatinase. It is a microbial enzyme which degrades creatinine in bacteria and provides source of carbon and nitrogen. Also, this enzyme is used in the enzymatic measurement of creatinine concentration for diagnosis of renal and muscles functions and diseases. Our purpose was recombinant production of creatinase for using in clinical measurement of serum or urine creatinine.

METHODS

A 1209bp of open reading frame of creatinase was amplified by PCR from genome and cloned into pET28a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET28a vector was transformed to . Creatinase expression was induced in using IPTG and confirmed by SDS-PAGE and western blotting. Purification of creatinase was performed using Ni-NTA column. The specific activity of this enzyme was also investigated.

RESULTS

The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 creatinase protein. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 that is much higher than other creatinase used in the commercial kits (9 ).

CONCLUSION

The recombinant creatinase was expressed efficiently in and 57% of total protein was the recombinant creatinase. Also, expressed creatinase has high solubility and also the enzyme has good activity compared to enzymes used in commercial kits, so a new source of creatinase was produced for creatinine assay kit in this study.