Fairlie W Douglas, Uboldi Alessandro D, De Souza David P, Hemmings George J, Nicola Nicos A, Baca Manuel
The Walter and Eliza Hall Institute of Medical Research, and The Cooperative Research Centre for Cellular Growth Factors, Post Office, Royal Melbourne Hospital, 3050, Parkville, Vic., Australia.
Protein Expr Purif. 2002 Oct;26(1):171-8. doi: 10.1016/s1046-5928(02)00521-1.
A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.
本文描述了一种用于生产、氧化和纯化含单个二硫键的短肽的重组融合蛋白系统。这些肽最初在大肠杆菌中表达，与细胞内磷酸酶SHP-2的N端SH2结构域的工程突变体融合。这个小蛋白结构域具有几个重要特性，有助于含二硫键肽的生产：（i）它在大肠杆菌中高水平表达；（ii）它可以通过六组氨酸标签和反相高效液相色谱法纯化；（iii）它不含内源性半胱氨酸残基，在仍与融合伴侣相连时允许形成肽内二硫键；（iv）它在天然缓冲液中高度可溶，便于生产非常疏水的肽，并直接在生化分析中使用融合产物；（v）它在肽和融合伴侣的连接处含有一个独特的甲硫氨酸残基，便于用溴化氰（CNBr）处理进行肽切割。该方法对于生产通过传统化学合成方法难以制备的肽很有用，通过制备一些源自噬菌体展示文库的含疏水二硫键的肽已证明了这一点。
