Study On The Structure Of PHA Protein Family Of The Cyanobacterium Synechocystis PCC6803

PHA(Polyhydroxyalkanoate) is a polyester intracellular inclusion which is widely present in a variety of microorganisms and other organisms. Because of the advantages of biodegradability, biocompatibility and high crystallinity characteristics, PHA becomes an environmentally friendly thermoplastic which can be completely degraded by microorganisms in nature. The excellent properties of PHA make it to be a hot topic, however, the structure of the key enzymes in the PHA synthesis way is not fully clear understood.This paper showed that pET28a-SpPhaA, pET28a-SpPhaB, pET28a-SpPhaC,pET28a-SpPhaE, pColdⅡ-SpPhaA and pColdⅡ-SpPhaC expression vectors were constructed successfully. Then we transformed the correct plasmid into E.coli BL21(DE3)competent cells. The conditions of cell culture were optimized for improving the expression of soluble protein and then the preliminary crystallization condition of SpPhaB was screened.The optimized condition for SpPhaB expression was determined to be: 37 °C, 0.1mmol/L IPTG, and the induction time was 7h. The optimized condition for SpPhaE expression was determined to be: 25 °C, 0.5 mmol/L IPTG, and the induction time was 7 h.After purifying by affinity chromatography with Ni column and FPLC, we can get 5 mg SpPhaB and 2.925 mg SpPhaE per liter bacterial. Hampton company crystallization kit was used for screening protein crystallization conditions. Mean the while, the protein concentration, sedimentation concentration and salt concentration was optimizting. Finally the optimized condition for SpPhaB crystallization was determined to be: 4 °C, 0.22 mol/L Magnesium acetate tetrahydrate, 0.1 mol/L sodium cacodylate trihydrate, pH 6.5, 18%PEG8000 crystalline medium, while mixing 1.5 μL protein in concentration of 10 g/L with1.5 μL crystal culture solution. The experimental results reveal the structure of the key enzyme in the PHA synthesis pathway and thus lay a foundation for understanding the binding mechanism of the enzyme and substrate.