使用通过定向进化获得的银河糖氧化酶的工程变体进行糖蛋白标签
Julie B Rannes1, Avgousta Ioannou, Simon C Willies
1School of Chemistry, University of Manchester, Manchester Interdisciplinary Biocentre, Manchester, M1 7DN, UK.
Journal of the American Chemical Society
|April 30, 2011
概括
研究人员设计了银河糖氧化酶变体,以选择性地修改糖蛋白上的甘氨酸. 这允许精确标记糖蛋白和整个细胞,在它们的表面上显示特定的糖.
科学领域:
- 生物化学 生物化学
- 酶学 是一种酶学.
- 葡萄糖生物学 葡萄糖生物学
背景情况:
- 葡萄糖蛋白在细胞过程中起着至关重要的作用.
- 选择性修改甘氨酸对于生物研究和治疗应用至关重要.
- 现有的糖甘标签方法在特异性或效率上可能受到限制.
研究的目的:
- 开发用于向氧化甘氨酸的新型酶变体.
- 为了允许选择性地在特定的甘氨酸上引入化物功能.
- 为了促进糖蛋白和细胞表面的后续标记.
主要方法:
- 使用定向进化来产生银河糖氧化酶 (GOase) 变体.
- 工程 GOase 变体的测试是为了测试它们氧化特定甘氨酸的能力.
- 引入的基组被评估为它们在标签上的有用性.
主要成果:
- 能够选择性氧化D-曼诺斯 (Man) 和D-N-乙葡萄糖胺 (GlcNAc) 甘的新型GOase变体已经成功生成.
- 酶变体在目标甘氨酸中引入了阿尔德海德功能.
- 这些基组被有效地用于标记改性糖蛋白和整个细胞.
结论:
- 定向进化为工程酶特异性提供了一个强大的策略.
- 开发的GOase变种提供了一个精确的工具,用于糖甘修饰和标签.
- 这种方法对化学生物学和细胞成像中的应用有很大的潜力.
相关概念视频
Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Oligosaccharide Assembly
Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
Multiple sugar molecules that may or may...
Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...


