甜味蛋白Brazzein基因的合成及在毕赤酵母中的表达

用连接PCR合成出甜味蛋白Brazzein的基因，克隆到PUC57质粒中测序后，用EcoR I和Not I双酶切，连接到pPIC9k，电转化毕赤酵母GS 115，经MD和MM平板筛选，获得His^ Mut^ 重组子。0．5％甲醇诱导表达96h后，对发酵液和细胞进行SDS—PAGE分析，在发酵液中没有蛋白带出现，在细胞裂解液中出现相对分子质量约6．0kd的特异性甜味蛋白条带，与预期的相对分子质量大小相符。用BCA蛋白定量试剂盒测定，Brazzein在毕赤酵母中的表达量可达329mg／L。

SYNTHESIS AND EXPRESSION OF BRAZZEIN GENE IN PICHIA PASTORIS

Brazzein gene was synthesized with PCR. After sequencing, the Brazzein-encoding gene was cloned into vector pPIC9k, which was then linarized with Sac1 and introduced into Pichia pastoris with the Gene Pulser System. After culturing on MD and MM medium plate, His~ Mut~s recombinants, which can stably express Brazzein, were obtained. Having been induced with 0.5 percent methanol for 96 h, the culture supernatant and the inclusion body were analyzed with SDS-PAGE. A band of 6.0 KD was shown migrating in the cell lysate on SDS-PAGE while no Brazzein was observed in the culture supernatant. The Brazzein concentration in the culture supernatant was as high as 329 mg/L in this study.