Cyclin A-Cdk2对B细胞成熟因子的体外磷酸化作用

In Vitro Phosphorylation of B Cell Maturation Factor with Cyclin A-Cdk2

目的原核表达并纯化仅含BH3结构域的细胞凋亡调控蛋白BMF,并进行CyclinA-Cdk2特异性底物的体外磷酸化检测。方法从HeLa细胞中扩增人源BMF基因,克隆至pGEX-6P-1表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经Glutathione Sepharose 4B凝胶柱亲和层析纯化。以纯化的融合蛋白GST-BMF作为底物,免疫共沉淀得到的CyclinA-Cdk2作为酶源,并以Cdk2的已知底物HistoneH1作为阳性对照,进行体外磷酸化试验。结果BMF基因的原核表达载体构建正确,表达的融合蛋白GST-BMF约占菌体总蛋白的40%,纯化后纯度约为90%。在体外磷酸化试验中,未出现与目的蛋白GST-BMF相对分子质量相近的放射自显影带。结论BMF蛋白在无细胞体系中不能被CyclinA-Cdk2磷酸化。

Objective To express the B cell maturation factor （BMF） only containing BH3 structural domain in prokaryotic cells, purify the expressed product for in vitro phosphorylation test and explore the possibility of BMF as a specific substrate of Cyclin A-Cdk2. Methods Amplify BMF gene from HeLa cells by PCR and insert into pGEX-6P-1 expression vector. Transform the constructed recombinant plasmid pGEX-6P-1-BMF to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed product was purified by Glutathione Sepharose 4B gel column affinity chromatography. Perform in vitro phosphorylation test using the expressed GST-BMF fusion protein as substrate, the Cyclin A-Cdk2 prepared by immuno-coprccipitation as enzyme source and the known Cdk2 substrate Histone H1 as positive control. Results The prokaryotic expression vector for BMF was constructed correctly. The expressed fusion protein GST-BMF contained about 40% of total somatic protein and reached a purity of about 90% after purification. No radioautographic band with similar relative molecular mass to that of GST-BMF was observed in in vitro phosphorylation test. Conclusion BMF could not be phosphorylated with Cyclin A-Cdk2 in a cell-free system.