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Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain

文献类型：期刊论文

作者Zou, Xiangman3,4; Liu, Zhi1,4,5; Song, Fengnan4,6,7; Zhou, Wei4,6,7; Hang, Jiaying4,7; Feng, Chenchen2,4; Yuan, Tianhong8; Dong, Jinhua3; Shi, Wei4,7; Tang, Feng3,4,6,7
刊名COMMUNICATIONS BIOLOGY
出版日期2025-08-06
卷号8期号:1页码:10
DOI10.1038/s42003-025-08614-7
英文摘要The removal of N-terminal methionine (Met) or fused N-terminal purification tags to expose the first amino acid of the target protein is critically important for studying the potential regulatory role of the N-terminal residue. However, current tag-removal approaches typically rely on enzymatic cleavage or require harsh reaction conditions. Here, we report a strategy for expressing proteins of interest (POI) with custom N-terminal amino acids by introducing an engineered cysteine protease domain (CPD) tag at the N-terminus. The cleavable tag is chemically triggered by inositol hexakisphosphate (InsP6), enabling precise generation of proteins with a user-defined N-terminus. Through systemic design and engineering of the N-terminal CPD tag, we successfully achieved POI variants with N-terminal Gly, Ser, His, Lys and other residues except Pro. In addition to the model protein, a Her2-targeting nanobody, we also successfully produced a RNF43-specific nanobody with an N-terminal Gln (N-Gln), an EGFR-targeting nanobody with N-Ala, the Sortase A enzyme with N-Gln, the fluorescent protein TurboGFP with N-Glu, and the sialic acid transferase Delta 15 Pd2,6ST with N-Cys. The construction of engineered nCPD-His10-POI further produced POI variants with a customized N-terminus and without any purified tags. Overall, the established approach enables high-yield protein expression and enzyme-independent, single-step removal of the redundant tag to yield proteins with the desired N-terminal residues, offering a valuable option for investigating N-terminal modifications and their functional implications.
WOS关键词AFFINITY PURIFICATION ; RECOMBINANT PROTEINS ; ESCHERICHIA-COLI ; FUSION PROTEINS ; CLEAVAGE ; ACTIVATION ; STRATEGIES ; PEPTIDE ; TAG
资助项目National Natural Science Foundation of China (National Science Foundation of China)[22422705] ; National Natural Science Foundation of China (National Science Foundation of China)[82325045] ; National Natural Science Foundation of China (National Science Foundation of China)[92478204] ; National Natural Science Foundation of China (National Science Foundation of China)[22337003] ; National Natural Science Foundation of China (National Science Foundation of China)[22277126] ; National Natural Science Foundation of China (National Science Foundation of China)[82204183] ; Natural Science Foundation of China (NSFC)[22YF1457400] ; Shanghai Sail Program ; SANOFI Scholarship Program
WOS研究方向Life Sciences & Biomedicine - Other Topics ; Science & Technology - Other Topics
语种英语
WOS记录号WOS:001546343300010
出版者NATURE PORTFOLIO
源URL[http://119.78.100.183/handle/2S10ELR8/321237]  
专题国家级研究中心_原创新药研究全国重点实验室
通讯作者Dong, Jinhua; Shi, Wei; Tang, Feng; Huang, Wei
作者单位1.Lingang Lab, Shanghai, Peoples R China
2.Henan Univ, Joint Natl Lab Antibody Drug Engn, Kaifeng, Peoples R China
3.Shenyang Pharmaceut Univ, Key Lab Struct Based Drug Design & Discovery, Minist Educ, Shenyang, Peoples R China
4.Chinese Acad Sci, State Key Lab Drug Res, Shanghai Inst Mat Med, Shanghai, Peoples R China
5.ShanghaiTech Univ, Sch Phys Sci & Technol, Shanghai, Peoples R China
6.Univ Chinese Acad Sci, Sch Pharmaceut Sci & Technol, Hangzhou Inst Adv Study, Hangzhou, Peoples R China
7.Univ Chinese Acad Sci, Beijing, Peoples R China
8.Guizhou Med Univ, Sch Basic Med, Guiyang, Guizhou, Peoples R China
推荐引用方式
GB/T 7714		 Zou, Xiangman,Liu, Zhi,Song, Fengnan,et al. Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain[J]. COMMUNICATIONS BIOLOGY,2025,8(1):10.
APA Zou, Xiangman.,Liu, Zhi.,Song, Fengnan.,Zhou, Wei.,Hang, Jiaying.,...&Huang, Wei.(2025).Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain.COMMUNICATIONS BIOLOGY,8(1),10.
MLA Zou, Xiangman,et al."Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain".COMMUNICATIONS BIOLOGY 8.1(2025):10.

入库方式： OAI收割

来源：上海药物研究所

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