复合干扰素在毕赤酵母中的表达与纯化

Expression and Purification of Consensus Interferon in Pichia pastoris

根据复合干扰素的氨基酸序列和毕赤酵母密码子偏爱性,利用重叠延伸PCR的方法合成复合干扰素DNA序列,克隆至分泌型酵母表达载体pGAPZαA,线性化后经高效电转化、Zeocin筛选鉴定及培养条件优化,获得了重组复合干扰素高表达菌株pGAP-conIFN/GS115,重组蛋白以可溶性分子的形式存在于上清液中.培养液上清经盐析、凝胶过滤和阴离子交换层析进行纯化,最后用分子筛脱盐.SDS-PAGE分析显示,纯化后的目标蛋白纯度可达到92%左右,比活性可达5.5×108U/mg.

To obtain high level secretive expressed con-IFN in Pichia pastoris, the DNA of con- IFN was amplified by recursive PCR, digested with EcoR I and Not I , then cloned into the secretory expression vector pGAPZaA. The recombinant vector was linearized, and transformed into GS115 through high efficiency transformation and Zeocin selection, the recombinant strains of pGAP-conIFN/GS115 were obtained. SDS-PAGE analysis suggested the recombinant protein was secreted into culture medium. The culture supernatant was purified through salt out, size exclude chromatography and ion exchange, the purity of recombinant protein reached 92%, purified recombinant con-IFN had a specific antiviral activity of about 5.5 × 10^8 U/mg.