人己糖胺酶D的原核表达、纯化及酶学特性研究

糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径.人己糖胺酶D( Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚.利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21( DE3) plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶.采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95％以上).以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2· 6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制.在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmoL/L,最大反应速率为3.06 μmol/( min·mg).

Expression,Purification and Enzymatic Characteristics of Human Hex D in E. coli

Glycosylation is an important protein post-translational modification,which is involved in many cellular processes,including signal transduction and cell recognition.Properly hydrolysis of glycoconjugates is essential for organism metabolism.Human hexosaminidase D(Hex D) is a newly discovered glycosidase which cleaves GalNAc(O-linked N-Acetyl-β-D-glatosamine) modification.However,the enzymatic characteristics of Hex D remains unknown.Here,the cDNA sequence was amplified by PCR and cloned into plasmid pET3C.After transformed the recombinant plasmids into Escherichia coli BL21(DE3) plys,the expression of Hex D was optimized with 0.1 mmol/L IPTG in 10 hours and purified it with the Ni-NTA affinity chromatography.SDS-PAGE verified the molecular weight(58 kDa) and the purity(> 95%).Using 4-MU-GalNAc(4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-galctopyranoside)as substrate,the optimal pH and temperature for Hex D is 5.5 and 37 ℃ respectively.Assay of Hex D pre-incubated for 30 min at different temperature indicated that it had high thermal stability and retain activity at 50 ℃.1mmol/L metal ions(CuSO4、FeSO4·7H2O、MgCl2·6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)and EDTA has no different effect on Hex D enzymic activity.However,10mmol/L AlCl3,CuSO4 or FeSO4·7H2O decreased the activity of Hex D to different extent.Using the optimum pH and temperature the Km value and Vmax of Hex D were 0.16mmol/L and 3.06 μmol/(min·mg) respectively.