重组人胸腺肽α1在大肠杆菌中的表达纯化与鉴定

Abstract

采用大肠杆菌作为宿主表达重组人胸腺肽α1（rhTα1）融合蛋白,纯化获得rhTα1并对其鉴定.于300L发酵罐进行中试发酵表达rhTα1融合蛋白;经异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导,离心发酵液收集菌体,菌体经破碎、离心、亲和层析捕获,获得融合蛋白;每升发酵液可获得约170 mg硫氧环蛋白-胸腺肽α1融合蛋白.融合蛋白经肠激酶酶切后,再经亲和层析、反相层析、体积排阻层析等纯化步骤,获得高纯度rhTα1;通过本纯化工艺获得的rhTα1原液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和高效液相色谱-排阻法（HPLC-SEC）的检测纯度均大于99%;采用玫瑰花环形成率测定rhTα1活性与市售化学合成标准品一致;质谱分析分子质量为3 066.59u,与去乙酰化的rhTα1理论分子质量（3 066u）一致;N端测序结果与理论值一致.综上结果,说明本工艺可实现工业化规模生产.In the present study,expression,purification and identification of recombinant human thymosin-alpha 1(rhTα1)in <i>Escherichia coli</i> were reported. Firstly,bacteria expressing the rhTα1 fusion protein were utilized in the 300 L fermentator,and protein was induced by IPTG.Then,bacteria were harvested by centrifugation,and cell lysate was obtained using high pressure crushes.The crude protein was purified using the affinity chromatograghy,and the yield of the fusion protein was up to 170 mg per 1 L culture. Furthermore,the crude protein was processed by enterokinase(EK)proteolysis,affinity chromatography,reversed phase chromatography and exclusion chromatography to obtain the high purity of rhTα1. SDS-PAGE and HPLC-RPC analysis indicated that rhTα1 could reach the purity of more than 99%. Additionally,rosette formation assay demonstrated that the activity of rhTα1 was quite similar to that of the commercial standard product. Molecular weight of rhTα1 by MALDI-TOF MS was 3 066.59 u,which agreed with the theoretic result(3 066 u),and the analysis of N-terminal of rhTα1 showed an accordance with the theoretic data,which all implied a potential for the industrialization of rhTα1.