Affinity Purification and Characterization of Recombinant Bacillus sphaericus Phenylalanine Dehydrogenase Produced by pET Expression Vector System

Author(s):

Eskandar Omidinia , Heshmatollah Taherkhani , Yasuhisa Asano , Shohreh Khathami , Alireza Omumi , Ata , Allah Ghadiri , Daniel Van Der Lelie , Roya Rashidpouraie , Hassan Mirzahoseini , Abbas Samadi

Message:
Abstract:
Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli weredone. The gene was cloned in the vector pET16b and transformed into E. coli BL21 (DE3). Thefunctional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purificationtechniques, 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 950mg cell pellet (wet weight). The Relative molecular mass of the L-phenylalanine subunits was about 41kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had aspecific activity of 577.3 U/mg protein, which is purified 88 times. This method was provided a facileand effective way for preparing the enzyme with a good yield that suitable for analytical purposes.
Language:
English
Published:
Iranian Biomedical Journal, Volume:6 Issue: 1, Jan 2002
Page:
31
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