人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达  

Expression of 2Gly-hGLP-1-HSA Fusion Protein in Pichia pastoris

作　　者：龙娟[1] 薛冲[1] 赵洪亮[1] 杨晓红[2] 刘志敏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]西南大学园艺园林学院,重庆400716

出　　处：《生物技术通讯》2008年第4期488-492,共5页Letters in Biotechnology

摘　　要：目的:在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1(hGLP-1)突变体(2Gly-hGLP-1)与人血清白蛋白(HSA)的融合蛋白。方法:为将GLP-1氨基酸序列第2位的丙氨酸(Ala)定点突变为甘氨酸(Gly),根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果:融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论:在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。Objective：To express fusion protein of mutant human glucogan like peptide-1（hGLP-1） and human serum albumin（HSA） in Pichia pastoris.Methods：The gene encoding 2Gly-hGLP-1,which is an analog of hGLP-1 with Ala substituted by Gly at position 2,was synthesized according to the prefered codon usage of P.pastoris.C-terminal of 2Gly-hGLP-1 and N-terminal of HSA was linked through a five-Gly spacer.The gene encoding 2Gly-hGLP-1-HSA fusion protein was obtained by overlap extension PCR and was cloned into pPIC9 vector.Then the expression vector of pPIC9/2Gly-hGLP-1-HSA was transformed into P.pastoris by electroporation.Through phenotype selection and expression assay,the expressing strain was obtained.After cultivation in a 5 L fermentor,the expressed products was purified and its bioactivity was analysed.Results：In a 5 L fermentor,the expression level of fusion protein reached 200 mg/L.After purification,the purity of 2Gly-hGLP-1-HSA exceeded 95%.The bioactivity analysis indicated that 2Gly-hGLP-1-HSA can reduce the inappropriate rise in glucose.Conclusion：These data demonstrated that 2Gly-hGLP-1-HSA expressed in P.pastoris can lower plasma glucose in vivo.

关 键 词：人胰高血糖素样肽 人血清白蛋白 融合蛋白 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]