Cloning And Expression Of Pyruvate Decarboxylase Gene

Pyruvate dehydrogenase complex is one of a-keto acid dehydrogenase complex family, which includs pyruvate decarboxylase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) and six cofactors. The function of pyruvate dehydrogenase complex is to catalyze the oxidative decarboxylation of pyruvate and produce acetyl-coenzyme A, NADH and CO2 in mitochondria. At the same time, the acetyl-coenzyme A is one of the important substrates in the TCA cycle and is oxidized to produce CO₂ and then provide energy to other physiological reaction. Only the first step of all oxidative actions is irreversible. If the corresponding inhibitors inhibit El, the cell cannot make use of oxygen. The accumulation of oxygen is harmful to the growth of organism, for example, it would disturb the construction of cell membrane;thus leading to organism death. However, the question on how to choose a high efficiency inhibitor from thousands of inhibitors cannot be solved. In this experiment, the recombinant expression system has been constructed to facilitate the preparation of a quantity of El with high purity and activity, so as to solve the question of shifting the pyruvate decarboxylase inhibitors with high activity.In this research, two different types of pyruvate decarboxylase genes, named a₂ and a₂β₂, have been successfully cloned and expressed.Firstly, a₂ from E.coli was cloned to vector pGEX-KG and the recombinant protein was obtained. As is well known, it's very difficult to produce the heterologous proteins of interest in the form of soluble body. Through the studies of pyruvate decarboxylase of E.coli, we successfully solved the key problem of inclusion body with SKL, which is a non-ion denature reagent. The results showed that with the treatment of SKL, pyruvate decarboxylase obtained in the soluble fraction exerted its activtity at 348U/ml, while without the treatment of SKL, the target protein was obtained in inclusion body and only showed the activity at 173U/ml after denaturation and renaturation.Secondly, as the sequence of rice pyruvate decarboxylase is unknown, we blasted rice genome.with the sequence of CDS of pyruvate decarboxylase from Arabidopsis thaliana. Then a sequence displayed more than 90% homology to Arabidopsis thalianaand we regarded it as the gene of rice pyruvate decarboxylase. This sequence was cloned into eukaryotic expression vector and then expressed in pichia pastoris to overproduce post-translationally modified protein of interest. Induced by methanol, the special protein has been yielded. The results from SDS-PAGE analysis provided further evidence for us that we have successfully cloned and expressed the a subunit and 8 subunit of pyruvate decarboxylase gene of rice, respectively. Furthermore, we have tred to assemble a subunit and B subunit in vitro to produce functional pyruvate decarboxylase. For the difficulty to assemble a functional pyruvate decarboxylase both with a subunit and B subunit in vitro without the help of molecular chaperone, we have not obtained a functional pyruvate decarboxylase yet.On the whole, the pyruvate decarboxylase from E.coli was gained, and this provided some conveience for the selection of corresponding inhibitors and also the researches on functional mechanism between them. At the same time, our researches on a subunit and B subunit of pyruvate decarboxylase of rice layed the foundation for further exploration into the struction and function of 8262 type of pyruvate decarboxylase.