Skerra A
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
J Biotechnol. 2001 Jun;74(4):257-75. doi: 10.1016/s1389-0352(01)00020-4.
The development of soluble receptor proteins that recognise given target molecules--ranging from small chemical compounds to macromolecular structures at a cell surface, for example--is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called 'anticalins' offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a beta-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants--termed anticalins--have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.
识别特定靶分子的可溶性受体蛋白的开发——例如，从小分子化合物到细胞表面的大分子结构——在生命科学和生物技术领域正变得越来越重要。在过去的一个世纪里，这个应用领域一直由抗体主导，传统上是通过动物免疫产生抗体，但最近也可以通过蛋白质工程方法获得。所谓的“抗钙素”提供了另一种类型的配体结合蛋白，它是在脂质运载蛋白的基础上构建的支架。这种蛋白质结构的核心元素是由八条反平行链组成的β-桶结构，在其开口端支撑着四个环。这些环形成了脂质运载蛋白的天然结合位点，可以通过广泛的氨基酸置换在体外重塑，从而产生新的结合特异性。胆绿素结合蛋白（BBP）被用作制备具有16个选择性诱变残基的随机文库的模型系统。利用细菌噬菌粒展示和菌落筛选技术，从该文库中筛选出了几种脂质运载蛋白变体——称为抗钙素——它们对荧光素或地高辛等化合物表现出结合活性。抗钙素对其指定的配体具有高亲和力和特异性，以及快速的结合动力学，因此它们的功能特性与抗体相似。然而，与抗体相比，它们具有几个优点，包括更小的尺寸、由单条多肽链组成，以及一组简单的四个高变环，可以在基因水平上轻松操作。除了作为靶标的半抗原化合物外，抗钙素如果随机文库设计得当，也应该能够识别大分子抗原。因此，它们不仅应作为生物分析目的的有价值试剂，而且在替代抗体用于医学治疗方面也可能具有潜力。
