人羧肽酶A1活性中心的毕赤酵母可溶表达

目的:应用毕赤酵母表达系统表达人羧肽酶A1(carboxypeptidase A1,CPA1)活性中心蛋白.方法:从pGEX-CPA1重组质粒中扩增出CPA1活性中心基因,亚克隆入酵母表达载体pPIC9K,酵母重组表达载体pPIC9K-CPA1active center电转化入毕赤酵母SMD1168,并进行营养缺陷筛选和G418抗性筛选,对高拷贝整合的重组酵母表达菌株诱导表达.结果:构建得到酵母融合重组表达载体pPIC9K-CPA1active center,经G418浓度梯度筛选得到串联整合16个重组表达载体的重组酵母表达菌株,诱导表达后得到CPA1活性中心蛋白.结论:在毕赤酵母中成功表达了CPA1活性中心,为进一步研究CPA1活性中心在抗体导向酶-前体药物疗法中的应用打下了基础.

Soluble expression of human carboxypeptidase A1 active center in pichia yeast

AIM: To express the human carboxypeptidase A1 (CPA1) active center protein in soluble form in pichia yeast. METHODS: Human CPA1 active center gene was amplified from pGEX-CPA1 recombinant plasmid and subcloned into pPIC9K yeast expression vector. Recombinant vector pPIC9K-CPA1 active center was transformed into pichia SMD1168 by electroporation. Positive recombinant yeast strains were screened through nutrition defect and G418 resistance. The integrated multicopy recombinant yeast strains were induced for expression. RESULTS: The yeast recombinant expression vector pPIC9K-CPA1 active center was constructed and recombinant yeast strains which integrated over 16 recombinant vector copies were obtained. The human CPA1 active center protein was expressed in soluble form in pichia yeast after screened with G418. CONCLUSION: Human CPA1 active center protein has been expressed successfully in pichia yeast, which will be useful for further research on ADEPT of prospate.