豹蛙抗瘤酶的毕赤酵母高效表达、纯化及活性测定

Expression, purification and characterization of recombinant onconase expressed in Pichia pastoris

摘要:

豹蛙抗瘤酶 (Onconase，ranpirnase，ONC) 对体内外多种肿瘤有很强的杀伤作用，是当前全球重点研究的100种新药之一，为获得高表达与高活性的重组豹蛙抗瘤酶 (Recombination onconase，rONC)，根据成熟ONC的cDNA序列和毕赤酵母密码子偏好性设计基因并提高其GC含量，分泌信号肽采用酵母α交配因子的pre肽，分别构建表达载体pPIC9/ONC、pPIC9K/ONC和pPICZα-A/ONC，并转染毕赤酵母X-33、GS115和SMD1168，筛选阳性克隆并进行诱导表达。在摇瓶规模筛选最佳载体-宿主组合及优化培养条件之后，进行10 L规模最优培养基的筛选，发酵产物经双水相萃取偶联G50凝胶层析分离纯化。结果表明，pPICZα-A /X-33/ONC组合表达量优于其他组合，且在pH 5.5、23 ℃条件下诱导7 d，最高表达量达到13 mg/L；在10 L规模条件下，rONC于pH 5.5的低盐基础培养基 (Lower basic salt medium，LBSM)、甲醇浓度0.25%条件下诱导7 d，最高表达量为180 mg/L；纯化后的rONC纯度≥95%，收率高于90%；生物活性检测发现，rONC在体外能杀伤多种癌细胞。初步建立了rONC的高效表达与纯化体系，为后续的功能和作用机理研究奠定了一定基础。

Abstract:

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor’s prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A /X-33/ONC combination under the condition of pH 5.5 and 23 °C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.