人源SPINK6在大肠杆菌中的表达优化

Optimization of human SPINK6 expression in Escherichia coli

丝氨酸蛋白酶抑制剂Kazal型6(serine protease inhibitor Kazal-type 6,SPINK6)蛋白是医学上的重要蛋白。为了高效制备具有生物活性的SPINK6蛋白,使用大肠杆菌表达系统重组表达SPINK6蛋白,经过纯化和酶切后测定了其生物活性,并对其表达条件进行了优化。结果表明,飞行时间质谱测得产物的分子量为6061.51 Da,通过此法成功制备获得了重组SPINK6蛋白,并且重组SPINK6蛋白对激肽释放酶相关肽酶5(kallikrein-related peptidase 5,KLK5)(K i=3.02±0.26 nmol/L)和KLK14(K i=1.85±0.05 nmol/L)有较强的抑制作用,具有生物活性。采用pE-SUMO3质粒为载体,在菌体OD 600=0.4时加入1 mmol/L异丙基-β-D硫代半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)诱导表达3 h,重组SPINK6蛋白获得最大表达量,1 L培养液中获得重组SPINK63.32 mg。研究结果可为SPINK6蛋白功能及应用研究提供操作性强的制备方法和稳定的蛋白来源。

Serine protease inhibitor Kazal-type 6(SPINK6)is a kind of medically important protein.In order to effectively prepare SPINK6 with biological activity,SPINK6 was expressed recombinantly through Escherichia coli expression system.Its biological activity was determined after purification and enzyme digestion,and the expression conditions were optimized.The results show that the relative molecular mass of the protein is 6061.51 Da measured by time of flight mass spectrometry,and the recombinant SPINK6 protein is therefore successfully prepared by this method.Moreover,the recombinant SPINK6 boasts high inhibitory effects against KLK5(K i=3.02±0.26 nmol/L)and KLK14(K i=1.85±0.05 nmol/L),thus being biologically active.Maximal recombinant SPINK6 production amounts to 3.32 mg/L achieved by using pE-SUMO3 as a vector and adding 1 mmol/L IPTG at thallus OD 600=0.4 for 3 h.This study optimizes the preparation method of SPINK6 with high operability,which might provide stable resources of SPINK6 for further focus on its functions and applications.