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首页> 外文期刊>Bioprocess and Biosystems Engineering >Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli
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Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli

机译：L-精氨酸对大肠杆菌中表达的赖氨酸标记的人胰岛素样生长因子1复性的影响

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摘要

Insulin-like growth factor 1 (IGF1), a therapeutic protein, is highly homologous to proinsulin in 3-dimensional structure. To highly express IGF1 in recombinant Escherichia coli, IGF1 was engineered to be fused with the 6-lysine tag and ubiquitin at its N-terminus (K6Ub-IGFl). Fed-batch fermentation of E. coli TGI/ pAPT-K6Ub-IGFl resulted in 60.8 g/L of dry cell mass, 18% of which was inclusion bodies composed of K6Ub-IGF1. Subsequent refolding processes were conducted using accumulated inclusion bodies. An environment of 50 mM bicine buffer (pH 8.5), 125 mM L-arginine, and 4 ℃ was chosen to optimize the refolding of K6Ub-IGFl, and 240 mg/L of denatured K6Ub-IGFl was refolded with a 32% yield. The positive effect of L-arginine on K6Ub-IGF1 refolding might be ascribed to preventing unfolded K6Ub-IGFl from undergoing self-aggregation and thus increasing its solubility. The simple dilution refolding, followed by cleavage of the fusion protein by site-specific UBP1 and chromatographic purification of IGF1, led production of authentic IGF1 with 97% purity and an 8.5% purification yield, starting from 500 mg of inclusion bodies composed of K6Ub-IGFl, as verified by various analytical tools, such as RP-HPLC, CD spectroscopy, MALDI-TOF mass spectrometry, and Western blotting. Thus, it was confirmed that L-arginine with an aggregation-protecting ability could be applied to the development of refolding processes for other inclusion body-derived proteins.
机译：胰岛素样生长因子1（IGF1）是一种治疗性蛋白质，在3维结构中与胰岛素原高度同源。为了在重组大肠杆菌中高表达IGF1，对IGF1进行了工程改造，使其在其N末端与6-赖氨酸标签和泛素融合（K6Ub-IGF1）。大肠杆菌TGI / pAPT-K6Ub-IGF1的分批补料发酵产生60.8g / L的干细胞团，其中18％是由K6Ub-IGF1组成的包涵体。随后的重折叠过程是使用累积的包涵体进行的。选择50 mM Bicine缓冲液（pH 8.5），125 mM L-精氨酸和4℃的环境优化K6Ub-IGF1的重折叠，并以240％/ L的变性K6Ub-IGF1重折叠。 L-精氨酸对K6Ub-IGF1重新折叠的积极作用可能归因于防止未折叠的K6Ub-IGF1经历自聚集并因此增加其溶解度。简单的稀释重折叠，然后通过位点特异性UBP1裂解融合蛋白并进行IGF1层析纯化，从500 mg包含K6Ub-的包涵体开始，可生产出纯度为97％，纯化率为8.5％的真实IGF1。经各种分析工具验证的IGF1，例如RP-HPLC，CD光谱，MALDI-TOF质谱和Western印迹。因此，证实了具有聚集保护能力的L-精氨酸可以用于开发其他包涵体来源的蛋白的重折叠过程。

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