PMID: 29090065Nov 2, 2017Paper

Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase

Avicenna Journal of Medical Biotechnology
Elnaz AfshariNahid Bakhtiari

Abstract

Pseudomonas putida (P. putida) 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. A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 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 Escherichia coli (E. coli) BL21 (DE3). Creatinase expression was induced in E.coli BL21 (DE3) 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. The creatinase gene cloning was confirmed by DNA sequencing. Successful expression of creatinase was performed in E. coli (57.4% of total protein). SDS-PAGE and western blot analysis showed a 45 kDa creatinase protein....Continue Reading

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