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首页> 外文期刊>Journal of Biotechnology >Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda P(L) promoter control: Temperature plays a major role in protein yields
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Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda P(L) promoter control: Temperature plays a major role in protein yields

机译：在细菌噬菌体λP（L）启动子控制下不同的人催乳素（hPRL）和生长激素（hGH）行为：温度在蛋白质产量中起主要作用

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摘要

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1mugml(-1)A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03mugml(-1)A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10mugml(-1)A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1mugml(-1)A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1IUmg(-1)).
机译：当生产用于治疗的重组蛋白时，不仅希望获得大量的蛋白，而且还希望确保最终产品中不存在潜在的污染物，例如诱导剂。为防止这种情况，可以使用这样的表达系统，其中启动子（lambdaP（L））通过使阻遏物（例如cIts）变性的温度变化激活。以这种方式，hGH在温度从30摄氏度转变为42摄氏度后在大肠杆菌周质中成功表达并分泌，比产量远高于1mugml（-1）A（600）（-1）。但是，尝试表达使用相同方案的相关激素人类催乳素失败，最多只能提供0.03mugml（-1）A（600）（-1）。描述了一种方法，其中在优化的温度条件（37摄氏度）下从cIts（-）菌株获得这种不稳定的蛋白质。因此获得了有史以来最高的催乳素周质分泌：0.92 +/- 0.10mugml（-1）A（600）（-1）在摇瓶培养中的光密度约为3 A（600）单位，光密度约为1mugml（通过实验室生物反应器中的快速灵活的分批进料过程，在35 A（600）单位的OD下获得-1）A（600）（-1）。纯化的hPRL是单体的，正确加工的（Mr = 22,906），正确折叠的且具有生物活性（51.5 +/- 24.1IUmg（-1））。

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