铜绿微囊藻气囊蛋白GvpW的异源表达纯化与晶体生长研究

Heterelogous expression,purification andcrystal growth of gas vesicle protein GvpW from the cyanobacterial Microcystis aeruginosa

铜绿微囊藻(Microcystisaeruginosa PCC 7806)是我国淡水湖泊生态系统季节性蓝藻水华爆发的最优势藻种,其爆发的机制之一在于气囊,它是一种充满气体的蛋白质性质的亚细胞器结构,可为藻细胞提供浮力,调节藻细胞在水体中的位置以利于生长和后续的生境殖民化。铜绿微囊藻的气囊合成涉及14个气囊蛋白,但大部分气囊蛋白在气囊合成过程中的具体分子功能尚不清楚。对其中一个气囊蛋白Gvp W进行了分子克隆,利用原核表达系统在体外进行了异源表达,结合镍柱亲和层析和凝胶过滤层析纯化出了高纯度的Gvp W,运用动态光散射和化学交联证实在体外纯化后的Gvp W是呈单体状态,同时通过晶体初筛和优化获得了生长较好的蛋白质晶体。这为后续解析Gvp W的三维结构及在分子水平直观探讨其在气囊合成中的生理功能奠定了基础。

Microcystis aeruginosa PCC 7806 is the most dominant algae of seasonal outbreak cyanobacteria bloom in fresh water lake ecosystem of China. One of the outbreak mechanisms for the occurrence of cyanobacterial water-bloom is gas vesicles, which are gas-filled proteinaceous organelles and provide varying buoyancy for cyanobacteria to regulate the position for growth and subsequent colonization. The gas vesicle of M. aeruginosa is composed of 14 gas vesicles proteins, but it is still unclear what the specific molecular functions of most Gvp proteins are during the process of gas vesicle synthesis. In this study, a gvpW gene was cloned and heterologous expressed in Escherichia coli, and the re- combinant protein was purified by Ni2~ affinity chromatography and gel filtration. Dynamic light scattering and chemi- cal crosslinking analyses showed that the recombinant protein GvpW mainly existed as monomer in vitro. In addition, GvpW protein crystals were obtained by crystal screening and optimization. These results provide a basis for further studies on its three-dimensional structure determination and molecular functions.