¦¡-ketoglutarate Dehydrogenase Complex Gene Cloning And Expression In E. Coli,

The α-ketogulatarate dehydrogenase is a key enzyme of the TCA cycle, which is composed by scores of subunits encoded by three genes. The three genes are sucA, sucB and lpdA. The sucA encodes α-ketogulatarate dehydrogenase, sucB encodes dihydrolipoyl transsuccinylase which contains covalently bound α-lipoyl groups, lpdA encodes flavoprotein which contains FAD and is designated E3. The molecular weights of the three enzymes are Mr 94,000, 47,000 and 54,000D, separately, and bounding by non covalent bond. The α-ketogulatarate dehydrogenase contains 12 α-ketogulatarate dehydrogenase chains, 24 dihydrolipoyl transsuccinylase chains and 12 flavoprotein chains. Experiments indicate that the transsuccinylase can bind about six α-ketogulatarate dehydrogenase dimmers and about 18 flavoprotein dimmers. However, the highest activity is obtained when only about six flavoprotein dimmers.The sequence of E.coli K12 genome was obtained by literature search. Primers were disignde based on the sequence. The sucAB and lpdA gene fragments were amplified by PCR. Restriction enzyme sites for EcoR I and Sal I were added to the 5' and 3' ends of sucAB gene, separately. Restriction enzyme sites for Sal I and Hind III were added to the 5' and 3' ends of lpdA gene, separately. The amplified two fragments encoding α-ketogulatarate dehydrogenase complex were inserted into plasmid pUC18, separately. After transforming competent E.coli JM109, The recombinants were screened on the selected MacConkey Agar plates. The recombimant plasmids, pUC18-sucAB and pUC18-lpdA were isolated, identified and sequenced.In order to construct a recombinant plamid with the three genes (sucA, sucB and lpdA) for α-ketogulatarate dehydrogenase connected in tandem, the sucAB fragments were obtained by cutting plasmid pUC18-sucAB with EcoR I and Sal I, and then ligated to plasmid pUC18-lpdA cuted with the same Sal I and EcoR I. As a result, the recombimant plasmid, pUC18-sucAB-lpdA was successfully constructed and identified with agrose gel electrophoresis.