Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, China.
Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Wenhai Road, Aoshanwei, Qingdao, China.
Glycobiology. 2018 Dec 1;28(12):949-957. doi: 10.1093/glycob/cwy077.
O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoa, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcylation. This study aimed to improve the production of highly O-GlcNAcylated recombinant proteins in Escherichia coli (E. coli). To achieve this goal, we constructed a bacterial artificial chromosome-based chloramphenicol-resistant expression vector co-expressing O-GlcNAc transferase (OGT) and key enzymes (phosphoglucose mutase, GlmM and N-acetylglucosamine-1-phosphate uridyltransferase, GlmU) of the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis pathway in E. coli, which can effectively increase the O-GlcNAcylation of the OGT target protein expressed by another vector. The results revealed that the expression of GlmM and GlmU increases the cellular concentration of UDP-GlcNAc in E. coli, which markedly enhanced the activity of the co-expressed OGT to its target proteins, such as H2B, p53 and TAB1. Altogether, we established a widely compatible E. coli expression system for producing highly O-GlcNAcylated protein, which could be used for modifying OGT target proteins expressed by almost any commercial expression vectors in E. coli. This new expression system provides possibility for investigating the roles of O-GlcNAcylation in the enzymatic activity, protein-protein interaction and structure of OGT target proteins.
O-GlcNAc ylation 是后生动物核质蛋白丝氨酸/苏氨酸残基上普遍存在且动态的翻译后修饰，在许多生理和病理过程中发挥着关键作用。但是，大多数蛋白质上的 O-GlcNAc ylation 通常是亚化学计量的，这阻碍了 O-GlcNAc ylation 的功能研究。本研究旨在提高大肠杆菌（E. coli）中高度 O-GlcNAc ylated 重组蛋白的产量。为了实现这一目标，我们构建了一个基于细菌人工染色体的氯霉素抗性表达载体，共表达 O-GlcNAc 转移酶（OGT）和关键酶（磷酸葡萄糖变位酶、GlmM 和 N-乙酰葡萄糖胺-1-磷酸尿苷转移酶、GlmU）的尿苷二磷酸 N-乙酰葡萄糖胺（UDP-GlcNAc）合成途径在 E. coli 中，这可以有效地增加另一个载体表达的 OGT 靶蛋白的 O-GlcNAc ylation。结果表明，GlmM 和 GlmU 的表达增加了 E. coli 中 UDP-GlcNAc 的细胞浓度，这显著增强了共表达的 OGT 对其靶蛋白（如 H2B、p53 和 TAB1）的活性。总之，我们建立了一个广泛兼容的大肠杆菌表达系统，用于生产高度 O-GlcNAc ylated 蛋白，该系统可用于修饰大肠杆菌中几乎任何商业表达载体表达的 OGT 靶蛋白。这个新的表达系统为研究 O-GlcNAc ylation 在 OGT 靶蛋白的酶活性、蛋白质-蛋白质相互作用和结构中的作用提供了可能性。
