ISSN: 0973-7510
E-ISSN: 2581-690X
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.
L-phenylalanine dehydrogenase, expression, plasmid, promoter
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