Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain
Abstract 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 enzym...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-08-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-08614-7 |
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| author | Xiangman Zou Zhi Liu Fengnan Song Wei Zhou Jiaying Hang Chenchen Feng Tianhong Yuan Jinhua Dong Wei Shi Feng Tang Wei Huang |
| author_facet | Xiangman Zou Zhi Liu Fengnan Song Wei Zhou Jiaying Hang Chenchen Feng Tianhong Yuan Jinhua Dong Wei Shi Feng Tang Wei Huang |
| author_sort | Xiangman Zou |
| collection | DOAJ |
| description | Abstract 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 Δ15 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. |
| format | Article |
| id | doaj-art-62e6d3f4d7ca4c71901b4df96a4458da |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-62e6d3f4d7ca4c71901b4df96a4458da2025-08-20T03:46:13ZengNature PortfolioCommunications Biology2399-36422025-08-018111010.1038/s42003-025-08614-7Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domainXiangman Zou0Zhi Liu1Fengnan Song2Wei Zhou3Jiaying Hang4Chenchen Feng5Tianhong Yuan6Jinhua Dong7Wei Shi8Feng Tang9Wei Huang10Key Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical UniversityState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesSchool of Basic Medicine, Guizhou Medical UniversityKey Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical UniversityState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of SciencesKey Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical UniversityKey Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical UniversityAbstract 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 Δ15 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.https://doi.org/10.1038/s42003-025-08614-7 |
| spellingShingle | Xiangman Zou Zhi Liu Fengnan Song Wei Zhou Jiaying Hang Chenchen Feng Tianhong Yuan Jinhua Dong Wei Shi Feng Tang Wei Huang Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain Communications Biology |
| title | Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain |
| title_full | Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain |
| title_fullStr | Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain |
| title_full_unstemmed | Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain |
| title_short | Facile expression of proteins with desired N-terminal amino acid via an engineered cysteine protease domain |
| title_sort | facile expression of proteins with desired n terminal amino acid via an engineered cysteine protease domain |
| url | https://doi.org/10.1038/s42003-025-08614-7 |
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