Ge Xin, Yang Daniel S C, Trabbic-Carlson Kimberly, Kim Bumjoon, Chilkoti Ashutosh, Filipe Carlos D M
Department of Chemical Engineering, McMaster University, 1280 Main Street West JHE-377, Hamilton, Ontario L8S 4L7, Canada.
J Am Chem Soc. 2005 Aug 17;127(32):11228-9. doi: 10.1021/ja0531125.
A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.
描述了一种纯化重组蛋白的简单方法，即将目标蛋白与一个内含肽和一个类弹性蛋白多肽融合，该方法仅需氯化钠、二硫苏糖醇和一个注射器滤器就能从大肠杆菌裂解物中分离出目标蛋白。这种三方融合系统能够快速分离目标蛋白，无需进行亲和层析纯化，也无需使用蛋白酶从融合蛋白中切割目标蛋白。类弹性蛋白多肽标签赋予三方融合体可逆的相变行为，从而通过添加氯化钠使融合蛋白在细胞裂解物中选择性聚集。通过微滤分离聚集体，并在低离子强度缓冲液中通过相变逆转使其重新溶解。在融合蛋白重新溶解后，激活内含肽以从弹性蛋白 - 内含肽标签上切割目标蛋白，然后通过额外的相变循环从弹性蛋白 - 内含肽融合体中分离出目标蛋白。
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