Horiya Satoru, Bailey Jennifer K, Krauss Isaac J
Brandeis University, Waltham, MA, United States.
Brandeis University, Waltham, MA, United States.
Methods Enzymol. 2017;597:83-141. doi: 10.1016/bs.mie.2017.06.029. Epub 2017 Aug 18.
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein. Although yeast, mammalian and Escherichia coli cells can be engineered to produce a homogenous glycoform at all glycosylation sites, there are just a few specific glycan structures that can readily be accessed in this manner. Recently, we reported a novel system for the directed evolution of glycopeptide libraries, which could in principle be decorated with any desired glycan. Our method combines in vitro peptide selection by mRNA display with unnatural amino acid incorporation and chemical attachment of synthetic oligosaccharides. Here, we provide an updated and optimized protocol for this method, which is designed to create glycopeptide mRNA display libraries containing ~10 sequences and select them for target binding. The target described here is the HIV broadly neutralizing monoclonal antibody 2G12; 2G12 binds to cluster of high-mannose oligosaccharides on the HIV envelope glycoprotein gp120; and glycopeptides that mimic this epitope may be useful in HIV vaccine applications. This method is expected to be readily applicable for other types of glycans and targets of interest in glycobiology.
定向进化是一种用于发现具有所需结合能力或功能的核酸、肽或蛋白质的有用方法。由于糖基化在自然界中的丰富性和重要性，糖肽和糖蛋白的定向进化也备受期待。然而，诸如噬菌体展示、酵母展示或哺乳动物细胞展示等常见的定向进化平台在这些应用中受到多种因素的限制。每个糖基化位点的聚糖结构并非由基因编码，并且酵母和哺乳动物细胞在蛋白质的每个位点都会产生糖型的异质混合物。尽管酵母、哺乳动物和大肠杆菌细胞可以经过工程改造在所有糖基化位点产生同质糖型，但通过这种方式能够轻易获得的特定聚糖结构却很少。最近，我们报道了一种用于糖肽文库定向进化的新系统，原则上该系统可以用任何所需的聚糖进行修饰。我们的方法将通过mRNA展示进行的体外肽选择与非天然氨基酸掺入以及合成寡糖的化学连接相结合。在此，我们提供了该方法的更新和优化方案，该方案旨在创建包含约10个序列的糖肽mRNA展示文库，并针对靶标结合对其进行筛选。这里描述的靶标是HIV广谱中和单克隆抗体2G12；2G12与HIV包膜糖蛋白gp120上的高甘露糖寡糖簇结合；模拟该表位的糖肽可能在HIV疫苗应用中有用。预计该方法可轻松应用于糖生物学中其他类型的聚糖和感兴趣的靶标。
