摘　要：线性泛素链连接方式特殊且被发现时间较短，目前商用的线性泛素链特异性抗体种类少且特异性仍有待提高。RHD1是一种木瓜蛋白酶样去泛素化酶，仅特异性识别线性泛素链。其主要酶活位点残基的第13位半胱氨酸（C13）突变，使得RHD1突变体失去去泛素化功能，但是与线性泛素链的结合活性水平仍不高。本研究在此突变体的基础上继续优化互作界面点突变，期望实现提升特异性结合活性。为此，本研究利用RHD1和线性双泛素蛋白复合物结构模型分析蛋白质间互作界面上氨基酸的理化性质，设计了RHD1突变体文库。本研究通过重叠延伸PCR法定点突变RHD1多肽链氨基酸，使用大肠杆菌BL21（DE3）菌株表达突变体重组蛋白，采用酶联免疫吸附法（ELISA）成功筛选到与线性四泛素蛋白结合活性显著提升的RHD1突变体，该突变体包含C13A与A92T点突变。该RHD1突变体有望被开发成为一种有助于研究线性泛素链的蛋白质配体工具，并且有巨大的潜力应用于临床诊断和开发相关治疗药物。
关键词：RHD1去泛素化酶；线性泛素链；定点突变；原核表达；酶联免疫吸附法
中图分类号：Q81                                   文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.05.006

Abstract: The special connection approaches of linear ubiquitin chain have been elucidated in recent years. Currently, the types of commercial linear ubiquitin chain specific antibodies are limited and their specificity still needs to be improved. RHD1 is a papain-like deubiquitinating enzyme that specifically recognizes and cleaves linear ubiquitin chains. The mutation at C13 in its enzymatic activity site results in the loss of deubiquitination function for the RHD1. Meanwhile, the RHD1 with this mutation still exhibits low binding activity towards linear ubiquitin chain. On the basis of this mutant, we optimized the mutations at interaction interface between RHD1 and Di-Ub (M1-linked) in order to enhance the specific binding activity of RHD1. In this study, we utilized the structural model of RHD1 and Di-Ub (M1-linked) complex to analyze the physical and chemical properties of all amino acids at the interaction interface and subsequently designed a RHD1 mutant library. The site-mutations in RHD1 variant polypeptides were generated by using overlap extension PCR method, and all recombinant variant proteins were expressed by using E. coli BL21 (DE3) strain. By employing the enzyme-linked immune sorbent assay (ELISA ) method for screening, we successfully identified a RHD1 variant harboring C13A and A92T mutations, which showed significantly increased affinity to Tetra-Ub (M1-linked). This RHD1 variant holds great potential as a valuable tool for studying linear ubiquitin chains, and exhibits promising application in clinical diagnostics and the development of related therapeutic agents.
Key words: RHD1 deubiquitinating enzymes; Tetra-Ub (M1-linked); site-specific mutation; prokaryotic expression; ELISA
（Acta Laser Biology Sinica, 2024, 33(5): 439-449）