Schmidt T G, Koepke J, Frank R, Skerra A
Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany.
J Mol Biol. 1996 Feb 9;255(5):753-66. doi: 10.1006/jmbi.1996.0061.
The Strep-tag is a selected nine-amino acid peptide (AWRHPQFGG) that displays intrinsic binding affinity towards streptavidin and has been used as an affinity tag for recombinant proteins. In order to elucidate the molecular mechanism underlying this type of artificial protein-peptide recognition, X-ray crystallographic analyses and binding measurements were carried out. The crystal structure of the complex between recombinant core streptavidin and the synthesized peptide was solved and refined at 1.7 A resolution (space group I4(1)22; unit cell dimensions a = b = 58.3 A, c = 176.9 A). The Strep-tag was bound at the same surface pocket where biotin, the natural ligand of streptavidin, gets complexed. The peptide backbone exhibited 3(10)-helical conformation, with eight of the residues involved in protein contacts. The C-terminal Gly-Gly moiety of the Strep-tag participated in a salt bridge to Arg84 of streptavidin with its free carboxylate group. This finding explained why the use of the Strep-tag in fusions with recombinant proteins was restricted to their carboxyl end. Employing a synthetic peptide spot assay, the variant Strep-tag II was screened, which did not have this limitation. The isomorphous crystal structure of its complex with streptavidin revealed that a glutamate side-chain provided the salt bridge in this case, with an otherwise almost unchanged mode of binding. Affinity constants between the peptides and streptavidin were measured by isothermal titration calorimetry. A value of 2.7 x 10(4) M-1 was determined for the Strep-tag peptide, and slightly tighter binding was seen when the Strep-tag was applied as part of a bacterially produced fusion protein. This affinity is significantly higher, compared with values previously reported for shorter streptavidin-binding peptides, and agrees well with the remarkable selectivity observed in recombinant protein purification applications.
链霉亲和素标签是一种选定的九氨基酸肽（AWRHPQFGG），它对链霉亲和素表现出内在结合亲和力，并已用作重组蛋白的亲和标签。为了阐明这种人工蛋白-肽识别的分子机制，进行了X射线晶体学分析和结合测量。重组核心链霉亲和素与合成肽之间复合物的晶体结构在1.7埃分辨率下解析并精修（空间群I4(1)22；晶胞参数a = b = 58.3埃，c = 176.9埃）。链霉亲和素标签结合在链霉亲和素天然配体生物素形成复合物的同一表面口袋处。肽主链呈现3(10)-螺旋构象，其中八个残基参与与蛋白质的接触。链霉亲和素标签的C末端甘氨酸-甘氨酸部分通过其游离羧基与链霉亲和素的Arg84形成盐桥。这一发现解释了为什么将链霉亲和素标签用于重组蛋白融合时仅限于其羧基末端。采用合成肽点分析筛选了变体链霉亲和素标签II，它没有这种限制。其与链霉亲和素复合物的同晶型晶体结构表明，在这种情况下谷氨酸侧链提供了盐桥，而结合模式几乎不变。通过等温滴定量热法测量肽与链霉亲和素之间的亲和常数。链霉亲和素标签肽的测定值为2.7×10(4) M-1，当链霉亲和素标签作为细菌产生的融合蛋白的一部分应用时，观察到结合稍紧密。与先前报道的较短链霉亲和素结合肽的值相比，这种亲和力显著更高，并且与重组蛋白纯化应用中观察到的显著选择性非常吻合。
