Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; Division of General Internal Medicine, Department of Medicine, University of Alberta, Edmonton, AB, Canada.
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
FEBS Lett. 2014 Jan 21;588(2):247-52. doi: 10.1016/j.febslet.2013.09.028. Epub 2013 Sep 27.
Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications.
如今，蛋白质通常通过使用可溶性增强融合标签进行过表达，这些标签允许亲和层析纯化，并通过位点特异性蛋白酶切割进行后续去除。在这篇综述中，我们提出了一种使用专门设计用于在不溶性包涵体中积累的融合伴侣来生产蛋白质的替代方法。该策略适用于大量生产短肽、天然无序蛋白质和可以在体外有效复性的蛋白质。现在有许多融合蛋白系统可用于不溶性表达：TrpLE、酮固醇异构酶、PurF 和 PagP 等。理想的融合伴侣能够有效地将各种靶蛋白导向包涵体，以高纯度的大量形式积累，并且可以在常用变性剂中轻易溶解和纯化。在变性条件下去除融合伴侣在生化上具有挑战性，需要苛刻的条件（例如，70%甲酸中的氰化溴），这可能导致不想要的蛋白质修饰。最近在金属离子催化的肽键切割方面的进展允许使用更温和的条件，一些涉及镍或钯的方法可能很快会出现在更多的生物应用中。
