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成熟单结构域“三叶因子”肽pNR-2/pS2 Cys58和pNR-2/pS2 Ser58的制备与比较

Production and comparison of mature single-domain 'trefoil' peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58.

作者信息

Chadwick M P, May F E, Westley B R

机构信息

Department of Pathology, University of Newcastle upon Tyne, Royal Victoria Infirmary, U.K.

出版信息

Biochem J. 1995 Jun 15;308 ( Pt 3)(Pt 3):1001-7. doi: 10.1042/bj3081001.

DOI:10.1042/bj3081001
PMID:8948462
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1136822/
Abstract

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the 'trefoil' family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

摘要

本文描述了富含半胱氨酸的分泌蛋白“三叶因子”家族单结构域成员重组成熟pNR-2/pS2的制备与纯化。通过离子交换色谱对重组pNR-2/pS2进行分析，结果显示其具有异质性。用含巯基的试剂处理后，异质性降低，这表明其是由成熟pNR-2/pS2中半胱氨酸残基的奇数数目所致，将额外三叶结构域的半胱氨酸残基Cys58突变为丝氨酸残基进一步证实了这一观点。在非变性条件下对重组pNR-2/pS2 Cys58和pNR-2/pS2 Ser58蛋白进行电泳，证实Ser58突变体的同质性更高，且表明大多数pNR-2/pS2 Ser58与培养的乳腺癌细胞分泌的pNR-2/pS2以单一条带形式共同迁移。用各种巯基反应性试剂处理重组pNR-2/pS2蛋白表明，半胱氨酸在产生与乳腺癌细胞分泌的pNR-2/pS2共同迁移的重组pNR-2/pS2方面最为有效。二硫苏糖醇似乎会使蛋白质变性，而谷胱甘肽相对无效。用半胱氨酸处理的pNR-2/pS2 Cys58和未处理的pNR-2/pS2 Ser58通过凝胶过滤测得的表观分子量与乳腺癌细胞分泌的pNR-2/pS2相同。这是关于重组成熟单结构域三叶肽生产的首次报道，应极大地有助于阐明pNR-2/pS2的结构和功能。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/213f7b73f489/biochemj00061-0298-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/0a8860f16943/biochemj00061-0294-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/1e5a708b00db/biochemj00061-0296-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/7c18c7812900/biochemj00061-0297-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/7ce1a9c1210f/biochemj00061-0297-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/2468a82f6015/biochemj00061-0298-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/213f7b73f489/biochemj00061-0298-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/0a8860f16943/biochemj00061-0294-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/1e5a708b00db/biochemj00061-0296-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/7c18c7812900/biochemj00061-0297-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/7ce1a9c1210f/biochemj00061-0297-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/2468a82f6015/biochemj00061-0298-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4ec6/1136822/213f7b73f489/biochemj00061-0298-b.jpg

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本文引用的文献

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Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.人肠三叶因子的鉴定。一种靶向顶端分泌的肽在杯状细胞中的特异性表达。
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Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease.炎症性肠病中胃肠道上皮细胞三叶肽基因的表达
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hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus.
三叶因子相互作用蛋白TFIZ1在正常胃黏膜细胞中优先结合三叶因子蛋白TFF1，但这些蛋白的共表达在胃癌中失调。
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The human trefoil peptide, TFF1, is present in different molecular forms that are intimately associated with mucus in normal stomach.人三叶肽TFF1以不同的分子形式存在，这些形式与正常胃中的黏液密切相关。
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