A self-cleavable sortase fusion for one-step purification of free recombinant proteins
A self-cleavable sortase fusion for one-step purification of free recombinant proteins
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DOI：
10.1016/j.pep.2004.06.013
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发表时间：
2004-09-01
影响因子：
1.6
通讯作者：
Mao, HY
中科院分区：
文献类型：
--
作者：
Mao, HY
已经开发了一种新的蛋白融合系统，以在单个亲和层析步骤中产生游离重组蛋白。融合中的关键组分是来自金黄色葡萄球菌的分选酶A（SrtAc）的催化核心，其以中等活性识别并切割LPXTG序列处的Thr Gly键。此处的融合物由N-末端HiS（6）标签、SrtAc和LPETG接头组成，随后是C-末端的目的蛋白。融合蛋白在大肠杆菌中表达，并通过固定化金属离子亲和层析（IMAC）纯化。然后，在Ca 2+和/或三甘氨酸的存在下，固定的融合物在LPETG位点处经历柱上SrtAc介导的切割。具有额外N-末端甘氨酸的靶蛋白从融合物中释放，而N-末端部分保持与柱结合。由于裂解酶SrtAc作为与靶蛋白的融合物共表达，因此纯化系统消除了外源蛋白水解。该纯化方法简单、稳健、廉价、省时，并且允许通过一步色谱法纯化游离重组蛋白。(C)2004年爱思唯尔公司All rights reserved.
A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal HiS(6) tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography. (C) 2004 Elsevier Inc. All rights reserved.