G蛋白偶联受体激酶5在大肠杆菌中的表达纯化

Expression and Purification of GRK5 in E.coli

通过RT-PCR扩增得到了人G蛋白偶联受体激酶5(GProtein-coupledreceptorki-nase,GPK5)的基因,将其克隆到表达载体pET-28a中,并将该质粒转入大肠杆菌BL21(DE3),在10μmol/L异丙基硫代-β-D-半乳糖苷(IPIG)诱导培养下表达,通过分离纯化,得到部分纯化的GRK5蛋白。体外活性测定表明,纯化后的GPK5能够特异性地磷酸化视紫红质。动力学常数Km=4.3μmol/L,Vmax=25nmol/(mg·min),与文献报道的从真核表达系统中纯化得到的GRK5相近。

Human G proteincoupled receptor kinase5(GRK5) was expressed and characteriged in E.coli. The human GRK5 cDNA gained by RTPCR was amplified and aloned into the expression vector pET28a. The expression vector was then transformed into E.coli BL21 (DE3), and the expression of GRK5 was induced at 22°C with the addition of 10 μmol/L IPTG. The enzyme was partially purified by a series of columnchromatography. Then the activity of GRK5 was assayed in vitro.The results suggested that GRK5 could specifically phosphorylated rhodopsin receptor in a lightdependent manner. The kinetic analysis of the enzyme revealed that its Km=4.3 μmol/L and Vmax=25 nmol/(mg·min),which were similar to that of GRK5 purified from eukaryotic expression systems.