ABSTRACT
Background: 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
Methods: A 1209bp of open reading frame of creatinase was amplified by PCR from P. putida ATCC12633 genome and cloned into pET[2]8a expression vector which was digested using NheI and XhoI restriction enzymes. Cloning was confirmed by colony PCR, double digestion analysis and sequencing. Recombinant pET[2]8a 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
Results: 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. Purification of creatinase was done with high purity. The specific activity of recombinant enzyme is 26.54 unit/mg that is much higher than other creatinase used in the commercial kits [9 unit/mg]
Conclusion: The P. putida ATCC12633 recombinant creatinase was expressed efficiently in E. coli BL21 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
ABSTRACT
Background: Pivotal roles of Nerve growth factor [NGF] in the development and survival of both neuronal and non-neuronal cells indicate its potential for the treatment of neurodegenerative diseases. However, investigation of NGF deficits in different diseases requires the availability of properly folded human beta-NGF. In previous studies bacterial expression of hNGF demonstrated the feasibility of its overproduction. However, known limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds were evident
Objectives: Here an optimized system was developed to overexpress the soluble NGF in E. coli
Materials and Methods: The gene encoding the beta subunit of mature hNGF was optimized based on E. coli codon preference and cloned into pET-32a expression vector providing His- and Trx- tags for detection and increasing the solubility of recombinant protein, respectively. The recombinant DNA was expressed in E. coli Origami [DE3], which enhances the correct formation of disulfide bonds in the cytoplasm of E. coli. Different culture conditions were evaluated to increase soluble expression of the target protein
Results: The highest soluble expression level was achieved when E. coli Origami [DE3] cells expressing NGF were grown at 30[degree]C in TB medium with 0.2 mM IPTG induction at OD[600nm] = 1 for 4 h
Conclusions: Our results indicated that the recombinant NGF was successfully expressed as a soluble form