Ebersbach Hilmar, Fiedler Erik, Scheuermann Tanja, Fiedler Markus, Stubbs Milton T, Reimann Carola, Proetzel Gabriele, Rudolph Rainer, Fiedler Ulrike
Scil Proteins GmbH, Heinrich Damerow Str. 1, 06120 Halle (Saale), Germany.
J Mol Biol. 2007 Sep 7;372(1):172-85. doi: 10.1016/j.jmb.2007.06.045. Epub 2007 Jun 22.
The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.
作为抗体替代物的新型结合蛋白概念已得到迅速发展，目前已准备好在广泛的应用中实际使用。基于具有理想特性的合适支架的替代结合蛋白是在体外从组合文库中筛选出来的。在这里，我们描述了一种方法，利用人γ-B-晶状体蛋白的β-折叠，通过对根据结构和序列分析选择的八个溶剂暴露氨基酸残基进行随机化，生成一个通用结合位点。已从噬菌体展示文库中分离出针对多种大小和结构差异很大的靶标的特定变体，即所谓的亲合素。分离出的亲合素变体可以在大肠杆菌中作为可溶性蛋白产生，并且具有高水平的热力学稳定性。人野生型γ-B-晶状体蛋白和一个选定的亲合素变体的晶体结构分别已确定为1.7埃和2.0埃分辨率。这两个分子的比较表明，人γ-B-晶状体蛋白能够耐受氨基酸交换而无主要结构变化。我们得出结论，本质上稳定且易于表达的γ-B-晶状体蛋白为生成新型结合分子提供了合适的框架。
