:: Volume 6, Issue 1 (1-2002) ::
IBJ 2002, 6(1): 31-36 Back to browse issues page
Affinity Purification and Characterization of Recombinant Bacillus sphaericus Phenylalanine Dehydrogenase Produced by pET Expression Vector System
Eskandar Omidinia , Heshmatollah Taherkhani , Yasuhisa Asano , Shohreh Khathami , Alireza Omumi , Ata-Allah Ghadiri , Daniel Van der Lelie , Roya Rashidpouraie , Hassan Mirzahoseini , Abbas Samadi
Abstract:  
Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 (DE3). The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Blue 4. Approximately 3 mg of highly purified recombinant enzyme was obtained from 950 mg cell pellet (wet weight). The Relative molecular mass of the L-phenylalanine subunits was about 41 kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had a specific activity of 577.3 U/mg protein, which is purified 88 times. This method was provided a facile and effective way for preparing the enzyme with a good yield that suitable for analytical purposes.
Keywords: Bacillus sphaericus, L-phenylalanine dehydrogenase (PheDH), affinity purification, expression
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Omidinia E, Taherkhani H, Asano Y, Khathami S, Omumi A, Ghadiri A, et al . Affinity Purification and Characterization of Recombinant Bacillus sphaericus Phenylalanine Dehydrogenase Produced by pET Expression Vector System. IBJ. 2002; 6 (1) :31-36
URL: http://ibj.pasteur.ac.ir/article-1-573-en.html


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