Gong Xiangyi, Ding Chongyang, Liu Liming, Wu Jing
Wuxi Medical School, Jiangnan University, Wuxi 214122, China.
Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):545-52.
The aim of this study was to further increase the yield of insulin precursor by Pichia pastoris.
For this, we transformed the expression vector pPICZalpha-IP into P. pastoris X-33 using electroporation and screened two mutant strains B4 and S6 on the YPD plate containing 100 microg/mL zeocin. Both could overexpress human insulin precursor. Taking B4 and S6 as start strains, we repeatedly transformed SacI linearizing pPICZalpha-IP into P. pastoris X-33 by electroporation, then screened a new mutant strain 2B4 (with 7 copies) on the 1000 microg/mL zeocin YPD plate.
After cultivation, the human insulin precursor yield of 2B4 strain was 2.7 fold higher than that of B7. Meanwhile, the cell growth was not inhibited. The target gene transcription level of 2B4 was 2 fold higher than that of B7 by real-time quantification PCR.
The strategy of combining resistance screening and repeated electroporation was efficient to increase the copy number of target gene, so as to facilitate higher transcription level and enhance objective recombinant protein yield.
本研究的目的是进一步提高毕赤酵母胰岛素前体的产量。
为此，我们使用电穿孔法将表达载体pPICZalpha-IP转化到毕赤酵母X-33中，并在含有100μg/mL博来霉素的YPD平板上筛选出两个突变菌株B4和S6。两者均能过表达人胰岛素前体。以B4和S6作为起始菌株，我们通过电穿孔法将SacI线性化的pPICZalpha-IP反复转化到毕赤酵母X-33中，然后在含有1000μg/mL博来霉素的YPD平板上筛选出一个新的突变菌株2B4（有7个拷贝）。
培养后，2B4菌株的人胰岛素前体产量比B7高2.7倍。同时，细胞生长未受抑制。通过实时定量PCR检测，2B4的靶基因转录水平比B7高2倍。
抗性筛选与反复电穿孔相结合的策略能有效增加靶基因的拷贝数，从而促进更高的转录水平并提高目标重组蛋白产量。
