Journal of Pure and Applied MicrobiologyVol. 7 No. 1

Potential role of promoter in production level of Bacillus sphaericus phenylalanine dehydrogenase in Escherchia coli expression system

Navid Nezafat1, Eskandar Omidinia1-2, Saeid Rahman Zadeh2, Saeed Heidari keshel3 and Sanam Zandian4

1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran. 3Proteomics research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4Department of Pharmacognosy, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Received on 24 July 2012 and accepted on 07 September 2012

 

ABSTRACT

In order to investigate the Potential role of promoter in production level of phenylalanine dehydrogenase in E. coli expression system this study was made. The L-phenylalanine dehydrogenase gene (pdh) was isolated from B. sphaericus. The pdh gene was cloned into two expression vectors; pET-23a and pPR37 differing in their promoter, which were T7 and lPR respectively. Expression of gene was induced by adding 1 mM (final concentration) of IPTG for vector carrying T7 promoter or by temperature shift from 30 to 42 ?C for vector carrying lPR. The highest rate of expression was observed in pETpdh/BL21(DE3)plysS construction. The level of expression of other construction was very low. PheDH was purified by Ni-NTA chromatography column. The relative molecular mass of PheDH subunit was about 41KDa by SDS-PAGE 10%. Applying this method for purification the specific activity of enzyme was 705 U/mg protein. The purity and amount of proteins were assessed by SDS-PAGE. Our polyhistidine-tag enzyme can be ideally used for the immobilization on a nickel coated microliter plate surface.

Keywords : L-phenylalanine dehydrogenase, expression, plasmid, promoter.