易于基因产物加工和快速纯化的融合表达载体  

Fusion Expression Vectors for the Recombinant Gene Products Processed Easily and Purified Rapidly by the Affinity Chromatography

作　　者：李伯良[1] 杨新颖[1] 江智红[1] 胡明[1] 梁镇和[1] 

机构地区：[1]中国科学院上海生物化学研究所,上海200031

出　　处：《生物工程学报》1994年第3期206-212,共7页Chinese Journal of Biotechnology

摘　　要：从Protein A基因酶切分离出编码完整的结合IgG的B、C区域(PABC)的相应DNA片段,克隆进噬菌体M13。通过人工合成寡聚核苷酸引物,突变修饰B、C区域内分别含有的羟胺裂解位点,变Asn-Gly为Asn-Ala。利用突变修饰后的PBACm编码基因片段,构建了一组含有不同阅读框架的融合蛋白表达载体。进一步分别重组克隆了含人工合成的人胰岛素样生长因子Ⅰ(IGF-Ⅰ)、人和牛生长激素释放因子(GRF)等基因的表达质粒,在大肠杆菌中高效表达出PABC-IGF-Ⅰ、PABC-hGRF、PABC-bGRF及其衍生型融合蛋白。经SDS-PAGE测定分析,每立升摇瓶的融合蛋白产量可达100mg以上。上述高效表达的PABC融合蛋白,通过固相亲和层析柱一步分离,获得SDS-PAGE鉴定为近均一分子量纯化的PABC融合表达产物。A DNA fragment encoding IgG-binding domain B, C (PABC) was separated from protein A gene, cloned into phage M13 and modified by oligo-directed mutagenesis at the hydroxylamine-cleaved site from Asn-Gly to Asn-Ala in domain B and C, respectively. The modified PABCm gene fragment was used to construct one set of fusion expression vectors in different reading frame. Some processing sequences such as those recognized by enterokinase, collagenase, thrombin, activated factor X and cleaved by the hydroxylamine, N-chlorosuccinimide etc. can be created in the fusion site. Using the above vectors, some fusion proteins such as PABCm-IGF-I, -hGRF, -bGRF and their derivatives were highly expressed in E. colt. The yield of fusion proteins is over 100mg per liter culture by analysis of SDS-PAGE. The PABC fusion proteins can be rapidly purified by the affinity chromatography with a IgG-sepharose 4B column.

关 键 词：蛋白A 点突变 融合表达 载体 

分 类 号：Q782[生物学—分子生物学]