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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2009, Vol. 29 Issue (06): 1-06    
研究报告     
基因重组胰高血糖素样肽-1衍生多肽的表达和产物纯化与鉴定
马义1|李弘剑2|周天鸿2
1. 暨南大学生物工程研究所
2. 暨南大学生命科学技术学院
Expression of Gene Recombinant GLP-1 Derived Polypeptide and Its Purification and Identification
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摘要:

为研究利用基因重组方法生产人胰高血糖素样肽-1(GLP-1)衍生多肽的最佳表达及纯化条件,选用大肠杆菌偏爱密码子,以含人GLP-1的质粒为模板,用PCR方法合成全长人GLP-1衍生多肽基因,并定向插入到高效表达载体pMFH中,用大肠杆菌BL21进行表达,融合蛋白经Ni-NTA柱纯化后,用C18 Sep-Pak 反相柱脱盐,然后融合蛋白经甲酸水解,水解产物经Ni-NTA柱和高效液相色谱(HPLC)纯化制备后,目的肽由质谱鉴定。 实验结果表明:利用载体pMFH在BL21中,GLP-1衍生物的最佳诱导表达温度为37℃、诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)的最佳浓度为0.6mmol/L,最佳诱导表达时间为6h;HPLC分析和制备GLP-1衍生物最佳条件为:流动相A(10% CNCH3∶90% H2O,0.1%TFA),流动相B(100% CNCH3,0.1% TFA),流速1ml/min,30 min线性梯度洗脱,B相至70%,检测波长280nm;质谱鉴定GLP-1衍生物的分子量为5.492kDa,与理论值相符合。在最佳表达及纯化条件下可得GLP-1衍生多肽的产量可达到11.6mg/L发酵产物,纯度≥98%。

关键词: null    
Abstract:

Abstract In order to study the optimal expression and purification condition of the GLP-1 derived polypeptide, by PCR technology synthetizing the gene of the GLP-1 derived polypeptide with preference codon of E.coli with the plasmid containing human wild-type GLP-1 gene. Expressed fusion proteins were purified and desalted with Ni-NTA column and C18 Sep-Pak column, respectively. After chemical cleavaged by formic acid hydroformicant, the hydrolysis products were purified with Ni-NTA column and HPLC. The target peptide was identified by mass spectrum. Experiment results showed in E.coli BL21 the optimal expression condition as follow:inducing temperature is 37℃, inducing time is 6h, and the concentration of the IPTG is 0.6mmol/L. The optimal chromatographic condition of getting HPLC as follow:mobile phase A (10% CNCH3∶90% H2O,0.1%TFA),mobile phase B(100% CNCH3,0.1% TFA),flow rate is 1ml/min, 30 min of the linear gradient elution, B phase reaches 70% and the detection wave length is 280nm. The molecular mass of the GLP-1 derived polypeptide is 5.492 kDa,through mass spectrum identification. The yield of GLP-1 derived polypeptide prepared with the optimal expression and purification condition may reach 11.6mg/L fermentation product and its purity is equal or greater than 98%.

收稿日期: 2008-10-21 出版日期: 2009-07-02
ZTFLH:  null  
基金资助:

广州市科技攻关计划项目(2006Z3-E4152);其它项目基金

通讯作者: 马义     E-mail: tlihj@163.com
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引用本文:

马义1,李弘剑2,周天鸿2. 基因重组胰高血糖素样肽-1衍生多肽的表达和产物纯化与鉴定[J]. 中国生物工程杂志, 2009, 29(06): 1-06.

MA Xi- Li-Hong-Jian- Zhou-Tian-Hong. Expression of Gene Recombinant GLP-1 Derived Polypeptide and Its Purification and Identification. China Biotechnology, 2009, 29(06): 1-06.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I06/1

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