rhBACE的克隆、表达纯化与活性测定  

Cloning,Expression and Activity Assay of rhBACE

作　　者：郭志刚[1] 张巍[1] 陈新园[1] 傅一工[1] 孙自勇[1] 刘建宁[1] 

机构地区：[1]南京大学分子医学研究所,南京210093

出　　处：《南京大学学报（自然科学版）》2004年第2期164-169,共6页Journal of Nanjing University（Natural Science）

基　　金：教育部重大科技项目 (0 0 -0 3 ) ;教育部博士点基金 (2 0 0 2 840 2 5)

摘　　要：将重组人酸性蛋白水解酶原 (rh_proBACE)基因克隆到原核表达载体 pET2 8a质粒中 ,构建了pET2 8a_rh_proBACE重组表达载体 ,并在大肠杆菌菌株Rosetta中进行表达 .包涵体中的表达产物溶于 6mol/L盐酸胍 ,经Ni_Sepharose亲和层析纯化后 ,得到高纯度的rh_proBACE蛋白 .将此蛋白在复性液中重新折叠后 ,于酸性条件 (pH 4.5)下激活 ,切去酶原序列 ,产生有活性的rhBACE蛋白 ,并利用人工合成多肽底物 (BAS_1 31 )Beta_site APP cleaving enzyme(BACE)is a membrane_bound aspartyl protease that cleaves amyloid precursor protein(APP)to generate the N terminus of amyloid beta peptide and plays a critical role in Alzheimer’s disease(AD).It is one of the most important target for Alzheimer’s disease drug development.BACE is expressed as a precursor protein containing protease,transmembrane and cytosolic domain.pro_rhBACE is a human derivative truncated without the amino_terminal signal peptide. In the present study,the gene fragment of pro_rhBACE was constructed into a prokaryotic expression vector pET_28a(+),inserted between the Nde I.Bpu1102I restriction enzyme sites.Proteins expressed in this vector have a6_histidine tail as an affinity handle.The expression plasmid was then transformed into the host cell strain,E.coli Rosseta.Transformed cells were cultured to log phase and induced by addition of1mmol/L IPTG.pro_rhBACE was highly expressed as insoluble inclusion bodies.The expression product(inclusion bodies)was dissolved in6mol/L guanidine hydrochloride.The supernatant was directly loaded to Ni_NTA Sepharose affinity chromatography column.pro_rhBACE was eluted by200mmol/L imidazole under denaturing conditions and was finally isolated with purity of>95%.SDS.PAGE showed that the molecular weight of pro_rhBACE was about50kD,consistant to the theoretical value calculated from the composition of amino acids.Purified pro_ rhBACE was then refolded and concentrated.It was self_cleaved into the active enzyme(rhBACE)in NaAc buffer at pH4.5.Enzyme activity assay showed that rhBACE cleaved the synthetic peptide at the APP beta site.In conclusion,using the method described above,the active rhBACE can be successfully prepared.

关 键 词：rhBACE 克隆 表达 纯化 活性测定 Β分泌酶 阿尔茨海默病 BACE rh-proBACE 

分 类 号：R749.16[医药卫生—神经病学与精神病学] Q78[医药卫生—临床医学]