Cloning and expression of aldehyde reductase in E.coli

BACKGROUND AND OBJECTIVESAldehyde reductase is an enzyme that catalyze the NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and also in prokaryotes like E.coli. they are attractive biocatalysts for industrial applications like in food, pharmaceutical industry and fine chemical industry. Objective of this project is to clone and express the gene that encodes the aldehyde reductase enzyme in E.coli.MATERIALS AND METHODSIn this project, the gene coding for the aldehyde reductase enzyme of E.coli has been amplified with specific primers(aldehyde reductase-Sal۱-R,aldehyde reductase-Nco۱-F). The sequence digested using restriction enzymes (SalI,NcoI) that was already designed in the primers .Then the PCR product cloned into the pET۲۶b-Yiat vector. The cloning of this fragment in the vector was confirmed by direct colony PCR And digestion with restriction enzymes SalI and NcoI. The recombinant vector was transformed into a suitable expression host (E. coli Rosetta (DE۳)) and the resulting colonies were analyzed for expression and activity.RESULTS AND DISCUSSIONThe cloning of the gene Encoding the aldehyde reductase enzyme fragment was confirmed by PCR and agarose gel electrophoresis. Restriction digestion pattern of recombined construct confirmed also. Colonies that were induced by ۱mM IPTG showed biological activity compared to the control sample. Cloning the aldehyde reductase gene under the T۷ promoter and in the correct expression frame resulted in its expression in the host bacteria. This expression confers the reducing activity to the host, which can reduce NAD to NADH in the recombinant host cell. Optimizing the expression of aldehyde reductase enzyme in this host using different culture conditions is in progress.CONCLUSIONAccording to the previous explanations about the role of aldehyde reductase in various industries, biologically production of this enzyme is of great importance considering its advantages, and more efforts and research should be done in the field of optimizing its performance.

کلیدواژه ها:

aldehyde reductase ، biocatalysis ، cloning ، Escherichia coli ، gene expression