Ssp dnaB蛋白质内含子介导的重组人脑钠素的制备

脑钠素(BNP)是临床治疗代偿失调性心衰竭的有效药物。将脑钠素与组氨酸标签(His-tag)以及具有自我剪切功能的Ssp dnaB微型蛋白质内含子进行融合表达。表达产物经Ni-Sepharose亲和层析及体外复性处理后,用CM_纤维素对复性产物进行了浓缩,并通过改变CM-纤维素柱内的pH及温度,诱导Ssp dnaB微型蛋白质内含子的剪切作用,使脑钠素从融合蛋白中释放并与载体蛋白(His-DnaB)分离,再经C4反相高效液相色谱法进一步纯化后,从每升培养液中获得了2.8mg纯度达97%的重组人脑钠素。体外活性测定结果表明,重组人脑钠素对兔胸主动脉条具有显著的血管舒张效应,其EC50为1.94×10-6mg/mL。

Use of Ssp dnaB Mini-intein as a Fusion Partner for Preparation of Recombinant Human Brain Natriuretic Peptide

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.