Prokaryotic expression and crystallization optimization of SpPhaB and SpPhaE from Synechocystis sp. PCC 6803

Abstract:

[Objective] To investigate the structure and function of PHB synthesis family protein, we cloned the phaB and phaE of Synechocystis sp. PCC 6803 and constructed prokaryotic expression vectors. The conditions of cell culture were optimized for improving the expression level of soluble protein and then the crystallization condition of SpPhaB was screened. [Methods] The phaB and phaE were cloned from Synechocystis sp. PCC 6803 by PCR and ligated to pET28a expression vector. To improve the expression of SpPhaB and SpPhaE, the culture conditions including IPTG concentration, temperature and induction time were studied. Ni-NTA resin was used to purify His-SpPhaB and His-SpPhaE protein, and initial crystal culture condition of SpPhaB was screened. [Results] The sequencing results showed that pET28a-SpPhaB and pET28a-SpPhaE expression vectors were constructed successfully. The optimized condition for SpPhaB expression was determined to be: induction at 37 °C using IPTG concentration of 0.1 mmol/L, the induction time was 7 h and the rotation speed was 220 r/min. The optimized condition for SpPhaE expression was determined to be: induction at 25 °C using IPTG concentration of 0.5 mmol/L, the induction time was 7 h and the rotation speed was 220 r/min. [Conclusion] The pET28a-SpPhaB and pET28a-SpPhaE were constructed successfully and the crystallization conditions of SpPhaB have been optimized. It provided the foundation for investigating the structure and function of SpPhaB.

Key words:SpPhaB, SpPhaE, Polyhydroxyalkanoate, Expression optimization, Crystal