Cloning, expression and purification of pseudomonas putida ATCC12633 Creatinase

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

Overexpression of recombinant human teriparatide, rhPTH [1-34] in Escherichia coli: an innovative gene fusion approach

Background: Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone [1-34] has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hPTH

Methods: Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to Escherichia coli [E. coli] beta-galactosidase [LacZ] gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH [1-34] gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in E. coli [DH5 alpha] cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 mM IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase

Results: By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting

Conclusion: In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial beta-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point [pI]. This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue

[Inhibition of ackA and pta genes using two specific antisense RNAs reduced acetate accumulation in batch fermentation of E. coli BL21 [DE3]]

Expression of foreign proteins in E. coli is normally inhibited by exogenous production of acetate. To overcome this problem, various strategies have been proposed and tested to reduce the extent of acetate accumulation. Although these strategies can improve the outcome, the implementation of their proposed techniques is not practical. Because to achieve optimal results, it requires extremely tight control conditions and the actual cost is very high. Furthermore, a simple knockout mutation of the target metabolic pathway would not be appropriate because the acetate pathway plays an important physiological role in E. coli. In this study, we employed an antisense RNA strategy as an elaborated metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, acetate kinase [ACK] and phosphotransacetylase [PTA]. The fragments of antisense cassette were cloned sequentially in pBluescriptsk+ and completed cassette subcloned in pLT10T3. The function of this cassette was evaluated with RT-PCR and ACK and PTA assay. The effect of cassette on cell physiology was monitored by determination of optical density, glucose consumption and acetate production. We found that the antisense method partially reduced mRNA levels of the target genes, lowered the concentration of acetate in culture media and increased growth rate and final cell density in antisense-regulated strain. This strategy could provide us with a useful, inexpensive and practical tool to achieve a large-scale protein production system