Binz H Kaspar, Amstutz Patrick, Kohl Andreas, Stumpp Michael T, Briand Christophe, Forrer Patrik, Grütter Markus G, Plückthun Andreas
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Nat Biotechnol. 2004 May;22(5):575-82. doi: 10.1038/nbt962. Epub 2004 Apr 18.
We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.
我们在此报告锚蛋白重复序列（AR）蛋白在体外针对特定高亲和力靶标结合的进化情况。采用共有设计策略，我们生成了具有不同重复数且结合表面多样化的AR蛋白组合文库。含有两个和三个重复序列且两侧有“封端重复序列”的文库，用于针对麦芽糖结合蛋白（MBP）和两种真核激酶的核糖体展示筛选。我们迅速富集了亲和力在低纳摩尔范围内的靶标特异性结合物，并以2.3埃分辨率确定了其中一种所选AR蛋白与MBP复合物的晶体结构。这种相互作用依赖于设计的AR蛋白的随机位置，并且与天然的异二聚体蛋白质 - 蛋白质相互作用相当。因此，我们的AR蛋白文库是结合分子的宝贵来源，并且由于设计的AR蛋白具有非常有利的生物物理特性，是抗体文库的有吸引力的替代物。
