含短C肽人胰岛素原类似物DesB^30在毕赤酵母中的表达及纯化  

Expression of Human Mini-proinsulin DesB^(30) in Pichia pastoris and Procedure for Purifying The Expression Product

作　　者：高剑坤[1] 蔡绍皙[1] 范开[2,3] 冯强 陈海蓉 张益 胡伟 杨应彬[1] 

机构地区：[1]重庆大学生物工程学院 [2]重庆工学院 [3]重庆富进生物医药公司重庆400041 [4]重庆富进生物医药公司

出　　处：《生物化学与生物物理进展》2008年第1期63-68,共6页Progress In Biochemistry and Biophysics

摘　　要：合成含短C肽AAK,并去掉B链第30位苏氨酸(T)的人胰岛素原类似物:HMPIDesB30(human mini-proinsulin des B30)的cDNA,将其插入大肠杆菌和酵母菌的穿梭质粒pPIC9K.用电转移的方法将重组质粒HMPIDesB30/pPIC9K转入甲醇酵母GS115.用含不同G418浓度的YPD平板筛选高拷贝重组子.经优化条件下的高密度发酵,发酵液用SIPI-40大孔树脂吸附,大孔吸附树脂洗脱液经SP柱进一步纯化后,再用10～20mmol/L Zn2+沉淀,能得到纯度为95%的HMPIDesB30.通过16.5%Tricine SDS-PAGE、HPLC和质谱分析,表达产物分子质量与理论分子质量相符,经高密度发酵,表达量可达1.0g/L.纯化回收率可达60%.说明人胰岛素原类似物HMPIDesB30能在甲醇酵母中高效分泌表达,并能通过经济有效的方法纯化.The cDNA of a human Mini-proinsulin （B^1-B^29）-AAK- （A^1-A^21） which named HMPIDesB^30 （human mini-proinsulin des B^30 was synthesized and inserted into the Escherichia coli-yeast shuttle vector pPIC9K. The constructed plasmid, HMPIDesB^30/pPIC9K was transformed into the GS115 cells of the methylotrophic yeast, Pichia pastoris, using electrotransformation. A transformant with a high copy number of the gene integrated into the chromosome was obtained by YPD plates which contains increasing concentrations of G418. After fed-batch fermentation, the protein was purified by macroporous resin, ion-exchange chromatography and precipitated with 10-20 mmol/L Zn^2＋. The purity of HMPIDesB^30 is 95%, the expression level reached 1.0 g/L, and the recovery rate of purification is about 60%. The purified protein was characterized by 16.5% Tricine SDS-PAGE, HPLC, and mass spectrometry, and the molecular mass of the expressed products is in accordance with the cumulated value. HMPIDesB^30 can be secretorily expressed by Pichia pastrois. The composition of the fermentation medium, the optimal condition of the fermentation and an effective method to purify the expression products from the culture were established.

关 键 词：表达系统 人胰岛素原 甲醇酵母 

分 类 号：Q51[生物学—生物化学] Q78