摘要
将人ｐ５３ 基因装入 Ｐｉｃｈｉａ 分泌型质粒ｐ Ｈ Ｉ Ｌ Ｓ１ 中，酶切线性化后电穿孔导入酵母细胞进行整合，经筛选得到一高表达ｐ５３ 蛋白的克隆。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 显示表达量约占分泌总量的３０ ％ 。 Ｅ Ｌ Ｉ Ｓ Ａ 验证重组人ｐ５３ 存在免疫学活性。在诱导时就降低 Ｐｉｃｈｉａ 酵母系统水解酶活力等方面进行优化，经 Ｆ Ｐ Ｌ Ｃ 分离纯化得到约２００ ｍ ｇ／ Ｌ 表达量。
Recombinant human p53 protein was produced in methylotrophic yeast Pichia pastoris here. Using Nco I and Bam HI, human p53 cDNA was inserted into the Pichia expression vector pHIL S1. The constructed plasmid, pHIL S2 p53, was linearized by Bg1 II, and then transformed into Pichia pastoris GS115(his4Mut) by electroporation. Through selection of Mut s phenotype and expressing clones, a high expression transformant was obtained. In addition, the influences of the initial pH during cultivation and the addition of protease inhibitors on p53 expression of the recombinant cells were investigated. The p53 were separated from the broth and purified by ultrafiltration and Mono Q FPLC. About 200mg of purified p53 with the immune competence under ELISA assay was produced from 1L broth.
出处
《生物工程学报》
CAS
CSCD
北大核心
1999年第4期477-481,共5页
Chinese Journal of Biotechnology
