Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai 201620, People's Republic of China.
Appl Microbiol Biotechnol. 2013 Jan;97(2):829-35. doi: 10.1007/s00253-012-4601-3. Epub 2012 Dec 5.
Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.
自剪切弹性蛋白样蛋白（ELP）标签为重组蛋白纯化提供了一种非常有前途的工具。使用这种方法，目标蛋白通过简单的 ELP 介导的沉淀步骤进行纯化，然后进行自切割并去除 ELP 标签。然而，遗憾的是，内含肽在蛋白表达过程中通常会经历一定程度的预切割，这会显著降低最终产量。在这项研究中，我们通过将内含肽分成两个 ELP 标记的片段来解决这个问题。每个片段本身都不能进行预切割，但当在体外组装时，组装的片段会迅速释放目标蛋白。其结果是对标签切割反应进行了非常严格的控制，同时结合了 ELP 纯化方法的简单性。使用该系统，我们成功地纯化了四种不同大小的目标蛋白，最终产量与我们最初的连续内含肽-ELP 系统相当或更高。此外，我们展示了一种简化的分段内含肽方法，其中表达标记内含肽片段的细胞在细胞裂解之前进行组合，允许这些片段在单个反应混合物中共同纯化。
