Cloning, Expression and Purification of Creatininase From Pseudomonas Pseudoalkaligene KF707 in E. coli.

Document Type: Article

Authors

1 Iranian research organization for science and technology (IROST)

2 Iranian Research Organization for Science and Technology, biotechnology department

Abstract

Creatinine amidohydrolase(EC 3.5.2.10) catalyzes the reversible conversion of creatinine to creatine. Creatininase in combination with other enzymes is used for detection of creatinine in serum and urine which is of significant value for detection of renal, muscular and thyroid functions. The aim of this study was to produce recombinant creatininase enzyme in E.coli expression system to use it in creatinine assay kit. The pseudomonas pseudoalkaligene KF707 creatininase gene has been optimized and synthesized already. Subsequently, it has been subcloned into the pET28 expression vector then the expression vector has been transformed into the BL21 (DE3) cell and induced by IPTG, afterwards the expression has been evaluated using SDS-PAGE and western blot. The recombinant protein has been purified by Ni-NTA agarose resins and enzyme activity has been analyzed. A sharp 29kDa protein band has been observed on SDS-PAGE and confirmed by western blot. More than 40% of E.coli total protein was recombinant creatinase, The recombinant enzyme was purified with approximately 100% yield. The enzyme activity analysis showed that recombinant enzyme has 14 unit/ml activity. Recombinant p.pseudoalkaligene KF707 creatininase was produced for the first time and its good production yield confirmed that E.coli was an efficient expression system for its production.

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