Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli
Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli
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DOI：
10.1016/j.pep.2006.01.014
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发表时间：
2006-07-01
影响因子：
1.6
通讯作者：
Hooper, Lora V.
中科院分区：
文献类型：
--
作者：
Cash, Heather L.;Whitham, Cecilia V.;Hooper, Lora V.
再生（Reg）家族包括与C型凝集素具有同源性的广泛的、多样化的蛋白质组。该家族的几个成员在正常情况下在胃肠道中高度表达，并且通常在炎性肠病中表现出表达增加。然而，对Reg蛋白的功能知之甚少。并且这些蛋白质的碳水化合物配体的特征很差。我们在这里报告的第一个表达和纯化的Reg蛋白使用细菌系统。小鼠RegIII γ和其人类对应物HIP/PAP在大肠杆菌中表达，导致聚集的重组蛋白的积累。这两种蛋白质均通过甘氨酸辅助的程序复性，并使用阳离子交换色谱进一步纯化。通过SDS-PAGE、N-末端测序和MALDI-TOF质谱确认纯化蛋白的身份。尺寸排阻色谱显示，这两种蛋白质存在的单体，和圆二色性表明，它们的二级结构表现出的P-链，这是典型的C-型凝集素的优势。最后，RegIII γ和人HIP/PAP都与甘露聚糖结合，但不与单体甘露糖结合，从而初步了解其碳水化合物配体。因此，这些研究为进一步分析人类和小鼠RegIII蛋白功能提供了必要的基础。(c)2006年爱思唯尔公司All rights reserved.
The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function. and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIII gamma and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of P-strands which is typical of C-type lectins. Finally, both RegIII gamma and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function. (c) 2006 Elsevier Inc. All rights reserved.