人源蛋白酪氨酸磷酸酶PTP1B功能域及其全酶的原核表达及活性比较  

Comparison of the Activity of Human Protein Tyrosine Phosphatase PTP1B Function Domain and Holoenzyme Gene Expression in Prokaryotic Expression

作　　者：庞晓斌[1,2] 谢欣梅[1] 李晓婷[1] 王守宝[2] 杜冠华[2] 

机构地区：[1]河南大学药物研究所,河南开封475004 [2]中国医学科学院&北京协和医学院药物研究所国家药物筛选中心,北京100050

出　　处：《河南大学学报（自然科学版）》2014年第2期196-201,共6页Journal of Henan University:Natural Science

基　　金：国家自然科学基金资助(81273652)

摘　　要：通过设计引物,利用RT-PCR从人肝癌细胞(huh-7)中克隆蛋白酪氨酸磷酸酶(PTP1B)全长及功能域PTPc的cDNA序列并连接到pGEM-T Vector载体上,测序正确的cDNA序列连接到表达载体pET-32a(+)上,分别在大肠杆菌Transetta(DE3)和BL21(DE3)菌株中稳定表达目标蛋白,经Ni 2+亲和柱纯化后目的蛋白达到了电泳纯,通过酶促动力学方法分析两种蛋白的体外活性.本实验成功表达了PTP1B和PTPc可溶性蛋白,并发现Transetta(DE3)菌株较BL21(DE3)有更高的蛋白表达能力;酶促动力学分析表明,PTP1B全酶的Vmax为16.13mmol·L-1·s-1,Km为0.94mmol/L;其功能域PTPc的Vmax为5.49mmol·L-1·s-1,Km为0.54mmol/L.表明PTP1B全酶的活性高于PTPc功能域的活性.This study is aimed to compare the activity of human protein tyrosine phosphatase （PTP1B） functional domain and holoenzyme. Total RNA was isolated from the human hepatoma cells （huh 7）. PTP1B and PTPc cDNA sequence were amplified by using RT-PCR. The cloning plasmid pGEM hPTP1B and pGEM-hPTPc were constructed and sequenced after retrieval of the amplification products, then the correct sequencing of the cDNA were subcloned into the expression vector pET-32a （ ＋ ） and transducted into E. coli Transetta （DE3） and BL21 （DE3） stably, respectively, induced with IPTG and expressed proteins were analyzed with SDS-PAGE and Western blot. The expressed products were purified through Ni affinity column, and the enzyme activities of two proteins showed that the protein has phosphatase activities by enzyme kinetics method in vitro. The soluble proteins of PTP1B and PTPc were successfully expressed, which were found that the E. coli Transetta （DE3） has a higher protein expression than BL21 （DE3）. The enzyme kinetic analysis showed that the Vmax of PTP1B and PTPc protein were 16.13 mmol· L-1· s-1 and 5.49 mmol · L-1 · s-1 , respectively. The K,,, of PTP1B and PTPc were 0.94 mmol/L and 0.54 mmol/L, respectively. It is easy to see that the activity of PTP1B protein was higher than PTPc.

关 键 词：PTP1B PTPc 蛋白表达 酶活性 

分 类 号：Q555.7[生物学—生物化学]