大鼠羧肽酶原B在毕赤酵母中的表达与培养条件优化

Optimized expression of rat procarboxypeptidase B in Pichia pastoris

目的:利用毕赤酵母表达系统,表达大鼠羧肽酶原B(procarboxypeptidaseB,proCPB)蛋白,同时探讨最优表达条件,为规模化发酵生产羧肽酶B奠定基础。方法:以RTPCR法从SD大鼠胰腺细胞中克隆了proCPB基因,将其插入pPIC9载体,转入毕赤酵母Gs115细胞,在甲醇的诱导下表达目的蛋白。结果与结论:首次实现了proCPB在毕赤酵母中的成功表达;通过表达条件的优化,使用BMGY(pH6.0)培养基,添加0.5%(体积比体积)的酪蛋白水解物和3mmol/L的PMSF,于28℃,每隔12h补加0.5%(体积比体积)的甲醇,重组酵母Gs115proCPB表达的重组蛋白可达500mg/L,表达的目的蛋白占总蛋白的94%,活化后的重组CPB相对分子质量为35.1×103,与理论值(35.2×103)极相近。活性测定表明,重组proCPB活化后的CPB的比活力可达110U/mg(CPB参照品为180U/mg)。

Objective: To express the procarboxypeptidase B in Pichia pastoris expression system for scaling up the production of procarboxypeptidase B, and to optimize the expression conditions.Methods:Total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with Xho I and EcoR I ,the fragment was inserted into pPIC9, giving rise to pPIC9-proCPB. After digestion with Sac I,the lined pPIC9-proCPB was transformed into P. pastoris strains Gs115 by PEG1000 and integrated into their genomes. Results and Conclusion: By the induction of methanol, recombinant proCPB was successfully expressed in P. pastoris for the first time, and could be secreted into the supernatant in the culture . After optimizing the expression conditions, a high production could be obtained when Gs115-proCPB was induced in BMGY(pH6.0) at 28℃ with addition of 0.5% casein hydolysate, 3 mmol/L PMSF and 0.5% methanol every 12 hours.The yield of recombinant protein reached 500 mg/L, accounting for 94% of total protein in the culture supernatant. Mass spectrometry analysis showed the mass of the recombinant CPB was 35.1×10~3, which is very close to the theoretical value 35.2×10~3. Comparing to the specific activity 180 U/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110 U/mg.