摘要
腺苷酸核糖基化因子1(ADP ribosylation factor 1,ARF1)是一种小G蛋白,负责调控细胞内的囊泡运输,从而影响细胞的生长发育.采用分子克隆的方法构建人类ARF1(hARF1)蛋白的重组质粒pET28a-hARF1,并在大肠杆菌(Escherichia coli)BL21(DE3)中表达纯化,随后利用荧光光谱法和分子对接方法研究hARF1蛋白与嘌呤核苷酸(GDP/ADP)之间的弱相互作用.研究结果表明,重组表达的hARF1蛋白分子质量22 859.29u,与理论值基本一致,其纯度大于95%,产率为5mg/L左右;GDP/ADP与hARF1蛋白弱相互作用的结合常数分别为0.022 69和0.007 71(μmol/L)-1,说明hARF1蛋白选择性地结合GDP,这与细胞内hARF1蛋白只结合GDP的结论一致.
ADP ribosylation factor 1（ADP ribosylation factor 1,ARF1） is a small GTP-binding protein,which is widely distributed in eukaryotic cells.ARF1 interacts with Golgi apparatus,plays the role of regulator of vesicle trafficking,and affects the development of various diseases.In this paper,the recombinant plasmid pET28a-hARF1 was constructed,and hARF1 protein was expressed in Escherichia coli BL21（DE3） and purified by affinity chromatograph and gel filtration.The results showed the molecular mass of hARF1 protein was 22 859.29 u,which was consistent with the theoretical value.The purity of hARF1 protein was above 95%,while the yield was about 5 mg/L.At the same time,the interactions between hARF1 and GDP/ADP were investigated by using fluorescence spectroscopy and MOE-Docking.It was found that hARF1 and GDP/ADP had non-covalent interactions,and the binding constants were 0.022 69 and 0.007 71（μmol/L）-1 respectively.It means that hARF1 could bind to GDP selectively.The above-mentioned results were useful for the further study on the structure and function of hARF1.
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第6期1053-1058,共6页
Journal of Xiamen University：Natural Science
基金
国家自然科学基金项目(20732004,20805037)
