2-脱氧核糖-5-磷酸醛缩酶在毕赤酵母中的分泌表达

Secretory expression of 2-desoxyribose-5-phosphate aldolase in Pichia pastoris

为实现2-脱氧核糖-5-磷酸醛缩酶基因(DERA)在毕赤酵母中分泌表达。通过PCR从E.coli BL21基因组中扩增获得DERA序列,并且与毕赤酵母分泌表达载体pPIC9K进行连接,构建得到重组质粒pPIC9K-DERA。重组载体经salⅠ单酶切线性化后电击转化至毕赤酵母GS115感受态细胞,用MD/MM平板筛选阳性株并在含G418的YDP培养基中筛选多拷贝插入菌株。经PCR鉴定获得一株插入pPIC9K-DERA的多拷贝嗜甲醇重组毕赤酵母菌株。用甲醇诱导该重组菌株,分泌得到具有活性的2-脱氧核糖-5-磷酸醛缩酶,且72 h时酶活为2.4 U·mg-1。结果表明,2-脱氧核糖-5-磷酸醛缩酶可实现在毕赤酵母中分泌表达。

It was designed to express 2-deoxyribose-5-phosphate aldolase（ DERA） in P. pastori. The DERA gene was cloned from E. coli BL21 through PCR,and then was ligated into the expression vector pPIC9K of P. Pichia. The recombinant plasmid pPIC9K-DERA was finally obtained,which was then linearized by salⅠand transformed into P. pichia by electroporation. Positive clone was were screened on MD / MM plate,and G148 resistant colonies with multicopy inserts were screened on YPD plates by increasing concentrations of G418. A multicopy recombinant methylotrophic P. pichia with inserted pPIC9K-DERA was obtained by PCR identification. Using methanol induction this recombinant strain,it secreted 2-deoxyribose-5-phosphate aldolase having an activity of 2. 4 U / mg after induction for 72h. The results show that eukaryotic secretory expression of the DERA can be performed in P. pastoris.