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A Journal on Biotechnology and Molecular Biology
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Minerva Biotecnologica 2016 March;28(1):12-8
Molecular cloning, characterization and bioinformatics analysis of CYP152A1 gene from Bacillus subtilis in Escherichia coli
Mohammad J. RAEE 1, 2, Samira MAGHAMI 1, 2, Mohammad B. GHOSHOON 1, 2, Milad MOHKAM 1, 2, Mozhdeh ZAMANI 1, 2, Narjes EBRAHIMI 1, 2, Younes GHASEMI 1, 2
1 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; 2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
BACKGROUND: The cytochrome P450s constitute a large family of heme enzymes, which play a key role in the oxidative transformation of endogenous and exogenous molecules. Among bacterial sources, CYP152A1 protein isolated from Bacillus subtilis (B. subtilis) would be a great candidate as biocatalyst for clinical and industrial applications. Therefore, the development of rapid, simple and effective production of recombinant CYP152A1 are not only of academic interest, but also of practical importance.
METHODS: For cloning and expression studies, the YbdT gene coding for CYP152A1 was amplified. The PCR product was digested and then ligated into the vector pET-15b. E. coli DE3 was transformed with the ligation product. Transformed cell was amplified with T7 primers by PCR. Gene expression was induced at 30°C by IPTG. The enzyme was purified by Ni-NTA Spin Columns and the result was shown by SDS-PAGE analysis.
RESULTS: According to the agarose gel electrophoresis, cell was cloned successfully. The DNA sequence alignments resulting from the BLAST search of ybdT gene from B. subtilis showed more than 99% sequence identity with other B. subtilis bacteria isolated from other regions of the world. But no significant differences were found when comparing amino acid sequences.
CONCLUSION: Here we reported the successful production of CYP152A1 by cloning the YbdT gene obtained from B. subtilis PTCC 1023 in to PET-15b expression vector in E. coli and impressive expression of the gene carrying recombinant plasmid under IPTG inducible T7 promoter. This recombinant protein can be produced for many applications such as biotransformation of special compounds.