Expression And Refolding Of Malate Dehydrogenase Inclusion Bodies

Malate Dehydrogenase (MDH, EC 1.1.1.37) is an essential metabolic enzyme in tricarboxylic acid cycle. It catalyzes reversible oxidation of L-malate to oxaloacetate with the concomitant reduction of NAD+. For its highly selectivity of 4-carbon dicarboxylic acid substrate, it can be used as biocatalysts in the production of chiral 2-hydroxyacid, which is important in pharmaceutical industry. Moreover, MDH has homo-dimer structure, which is often used as a model to study oligomeric protein structure and function relationship. In this study, E.coli MDH (eMDH) was cloned, expressed in Escherichia coli (E.coli) but as inclusion bodies(IBs), and then how to regain eMDH activity from IBs was studied.Firstly, the gene encoding eMDH was amplified from chromosome of E.coli DH5αby polymerase chain reaction (PCR).Then the PCR fragment was cloned into the expression vector pBV220 to get recombinant plasmid. The positive recombinant, which was verified by restriction endonuclease digestion and sequence analyse, was transformed into host E.coli DH5α. The engineering strain was induced at 42oC, foreign protein eMDH was over-expressed in E.coli with a yield of 42%, mainly as IBs assayed by SDS-PAGE. The eMDH production can reach 87.3 mg?(L culture)-1. Subsequently, eMDH IBs were washed by Urea and Triton, with an enhancement in purity from 42% to 72%.Artificial chaperone (CTAB andβ-CD system) was applied in the refolding of eMDH IBs. The results indicated that artificial chaperone can assist eMDH IBs refolding effectively with a specific activity of 160U?mg-1, with the optimal conditions of CTAB:β-CD equal to 0.6mmol?L-1:4.8 mmol?L-1(1:8), 0.32 mol?L-1 of Urea, 5mmol?L-1 of DTT, pH7.6 at an enzyme concentration of 0.1mg?mL-1.Refolded eMDH was analyzed by fluorescence and circular dichroism (CD) spectroscopy, which showed that eMDH refolding assisted by artificial chaperone had more correct quaternary and secondary structures than that of dilution. Moreover, more dimer eMDH was gained by artificial chaperone refolding than dilution...