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首页> 外文期刊>Journal of Biotechnology >Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein
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Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

机译：利用麦芽糖结合蛋白的多功能性，利用大肠杆菌进行细胞外生产和重组蛋白的亲和纯化

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摘要

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to alpha -1,4-glucans.
机译：重组蛋白是当今工业生物技术的重要产品。在这项研究中，我们解决了重组蛋白生产中的两个关键因素：（i）产品可及性和（ii）产品回收率。大肠杆菌是重组蛋白表达最常用的宿主之一，它本身不会将蛋白分泌到细胞外环境中。因此，该表达系统的主要缺点在于细胞内蛋白质的积累和产物可及性的阻碍。我们构建了一组表达载体，以促进细胞外蛋白的产生和纯化。来自大肠杆菌的麦芽糖结合蛋白被用作几种目的蛋白的融合伴侣，从而允许通过Sec和Tat途径输出到细菌的周质。共表达修饰的Cloacin DF13细菌素释放蛋白后，杂合蛋白被释放到培养基中。这本质上适用于杰出的报告分子，绿色荧光蛋白，迄今为止尚未报道其胞外产生。可以通过利用麦芽糖结合蛋白对α-1，4-葡聚糖的亲和力的简单，快速和廉价的方法将螯合的蛋白纯化至近似均质。

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