摘要
将构建的含有胸腺肽α1融合蛋白的编码基因插入表达质粒pET28a(+)中,并在大肠杆菌BL21中进行表达,该菌经IPTG诱导融合蛋白高表达后,离心收集菌体,超声波破碎,包含体裂解,目标蛋白质的体外变性复性后,经DEAE-Sepharose Fast Flow阴离子交换层析纯化,每升菌液中可获得260 mg融合蛋白。经重组肠激酶酶切反应、Ni-Sepharose亲和层析,Sephadex G-25柱纯化后,得到了纯度达97%的重组人胸腺肽α1,该纯化工艺简单快速,经三步层析即可得到纯度达97%的重组人胸腺肽α1,且产量达82 mg/L,为大批量用细菌表达重组人胸腺肽α1及纯化提供了参考。
Human thymosin α1, a small hormone with a length of 28 amino acids, is a powerful antiviral drug for treatment of HBV, SARS and various cancers. After consideration of the feasibility of producing human thymosin α1 on a large scale, an over-expression vector has been constructed based on the plasmid pET28a（ ＋ ）. After induction by isopropyl β-D-1-thiogalactopyranoside （IPTG）, the observed yield of crude fusion proteins was up to 260mg/L. In order to obtain the purified target proteins, three different chromatographic columns, namely DEAE-Sepharose FF, Ni-chelating-Sepharose and Sephadex G-25, were operated in series. The purity of the resulting rhThymosin at was more than 97%, as shown by RP-HPLC analysis. Finally, 82 mg of purified rhThymosin α1 was obtained from α1 liter culture.
出处
《北京化工大学学报（自然科学版）》
CAS
CSCD
北大核心
2007年第3期296-299,共4页
Journal of Beijing University of Chemical Technology(Natural Science Edition)
