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Production of human pro-relaxin H2 in the yeast Pichia pastoris

机译：在酵母毕赤酵母中生产人类前松弛素H2

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BackgroundInitially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18?kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction. ResultsTo overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22?L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity. ConclusionsThis work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14?mg/L of product, as determined by densitometric analyses.
机译：背景技术松弛素最初被称为生殖激素，被证明具有其他治疗有用的特性，包括细胞外基质重塑，抗炎，抗缺血和血管生成作用。所有这些发现使松弛素成为多种医学应用的潜在药物。它的前体前松弛素是一种18kkDa的蛋白质，在体外测定中显示出活性。由于从动物组织中提取松弛素引起了许多问题，原核生物和真核生物都被用作重组松弛素生产的表达系统。当使用大肠杆菌作为人类松弛素表达的宿主时，可获得最有成果的结果。然而，在这种宿主中，松弛素以包涵体的形式沉淀，因此，需要几个昂贵的回收步骤，如细胞裂解，重折叠和还原。结果为了克服与原核表达有关的问题，我们在此报道了使用甲基营养酵母巴斯德毕赤酵母作为表达宿主来生产和纯化分泌的人类前松弛素H2。甲醇诱导型启动子AOX1在22？L规模的双阶段补料分批实验中用于驱动天然的和组氨酸标记的形式的前松弛素H2。两种蛋白质形式均表现出正确的结构，如通过质谱和Western印迹分析所确定的，并且在免疫酶分析中具有生物学活性。标签的存在允许简化松弛素原的纯化以获得更高的纯度。结论这项工作提出了一种在巴斯德毕赤酵母中微生物生产重组人前松弛素H2的策略，该策略允许获得具有生物活性的激素，最终发酵液中的终浓度为10至14？mg / L产品。通过光密度分析确定。

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