胸腺素家族多肽在大肠杆菌中的表达

Recombinant Expression of Thymosin Family Peptides in Escherichia coli

为提高胸腺素(又名胸腺肽,thymosin)的体外表达效率,本研究采用设计融合标签的方法对胸腺素α1和β4 (Tα1,Tβ4)进行体外重组表达,通过超高效液相色谱-飞行时间质谱联用(LC/MS)法,对融合蛋白的完整分子量进行验证,利用反相超高效液相色谱法对不同表达载体的单位表达量进行测定。结果表明,A18-KEKE、DKL6K两种融合标签能够高效地表达Tα1和Tβ4。LC/MS结果表明,所有被测样品的过表达条带中均含有融合标签的目的多肽,且带有融合标签A18-KEKE的胸腺素的单位表达量达73~123 mg·OD^(-1)·L^(-1)。A18-KEKE、DKL6K 2种融合标签可以有效地促进胰高血糖素样肽-1 (GLP-1)、胰高血糖素样肽-2 (GLP-2)、甲状旁腺激素(PTH 1-34)和糖依赖性胰样多肽(GIP)的表达,证实了这2种融合标签的广谱适用性。本研究成功筛选到了2种融合标签,可用于多种胸腺素家族多肽在大肠杆菌中的重组表达,且融合蛋白单位表达量较高,提高了胸腺肽类产品重组生产的效率,极大地降低生产成本,具有广泛的应用前景。

To improve the expression efficiency of thymosin, fusion tags were designed to perform recombinant expression of thymosin α1 and β4(Tα1, Tβ4) in vitro. The liquid chromatograph-mass spectrometer(LC/MS) method was used to confirm the complete molecular weight of fusion proteins with different expression vectors, and expression of different expression vectors was detected by reversed-phase ultra-high performance liquid chromatography. The results showed that two tags A18-KEKE and DKL6 K could efficiently express Tα1 and Tβ4. The LC/MS analysis showed that the over-expression bands of all tested samples contained the target peptides with the fusion tag A18-KEKE. The expression of thymosin was as high as 73-123 mg ·OD^(-1)·L^(-1). In addition, A18-KEKE and DKL6 K could effectively promote the expression of glucagon-like peptide-1(GLP-1), glucagon-like peptide-2(GLP-2), parathyroid hormone(PTH 1-34) and glucose-dependent insulinotropic polypeptide(GIP), confirming that the broad-spectrum applicability of these two fusion tags. In this study, we successfully screened two tags that can be used for the recombinant expression of different thymosin family polypeptides in E. coli, and the high expression levels of the fusion proteins indicates their prospects in industrial application prospect.