人α降钙素基因相关肽基因在酵母细胞中的分泌表达与产物的分离纯化

Secretory Expression and Purification of Human a Calcitonin Gene-related Peptide in Saccharomyces cerevisiae

化学合成的人α降钙素基因相关肽(CGRP)基因用PCR法改造后使其能正确融合在酵母分泌型表达载体pVT102U/α中的α交配因子前导肽序列之后,然后进行克隆并转化酵母宿主菌S-78进行表达,培养物的上清用酶标(ELISA)鉴定为阳性,而对照S-78、pVT102U/α为阴性,表达量用ELISA定量大于2mm/L。表达产物经阳离子交换层析(CM-Sephadex C_(25))和HPLC纯化得到了HPLC纯产品。纯化后的CGRP能引起小鼠血压的降低,说明表达的目的蛋白既有CGRP的免疫结合活性,又有CGRP的生理活性。测定其N-端10个氨基酸序列,证明人工合成的CGRP基因在酵母细胞中已正确表达。

A hybrid gene was constructed via polymerase chain reaction containing a fusion between the DNA sequences encoding the yeast mating pheromone a-factor leader region and a synthetic sequence encoding human a calcitonin gene-related peptide (haCGRP). Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active haCGRP into the extracellular medium. The expression level of haCGRP is more than 2mg/L. The secreted haCGRP was purified and found to be accurately processed at the junction in the chimeric a factor leader region/ haCGRP protein, producing the desired mature haCGRP amino terminus.