Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes
Abstract Background The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult. Results We introduce a working pipeline for expression, purification a...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-024-02316-1 |
| _version_ | 1797272216395579392 |
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| author | Jiang Xu Xiao Lei Ao Li Jun Li Shuxing Li Lin Chen |
| author_facet | Jiang Xu Xiao Lei Ao Li Jun Li Shuxing Li Lin Chen |
| author_sort | Jiang Xu |
| collection | DOAJ |
| description | Abstract Background The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult. Results We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. Conclusions Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications. |
| first_indexed | 2024-03-07T14:25:10Z |
| format | Article |
| id | doaj.art-60389c4a8f0643e8bda72292a47db618 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-07T14:25:10Z |
| publishDate | 2024-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-60389c4a8f0643e8bda72292a47db6182024-03-06T08:06:30ZengBMCMicrobial Cell Factories1475-28592024-02-0123111410.1186/s12934-024-02316-1Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probesJiang Xu0Xiao Lei1Ao Li2Jun Li3Shuxing Li4Lin Chen5Molecular and Computational Biology, Department of Biological Sciences, University of Southern CaliforniaMolecular and Computational Biology, Department of Biological Sciences, University of Southern CaliforniaMolecular and Computational Biology, Department of Biological Sciences, University of Southern CaliforniaInstitute of Clinical Medicine, The First Affiliated Hospital of University of South ChinaMolecular and Computational Biology, Department of Biological Sciences, University of Southern CaliforniaMolecular and Computational Biology, Department of Biological Sciences, University of Southern CaliforniaAbstract Background The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult. Results We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. Conclusions Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications.https://doi.org/10.1186/s12934-024-02316-1Three finger proteinE. coli recombinant expressionInclusion body refoldingDisulfide bond formationOxidation refolding |
| spellingShingle | Jiang Xu Xiao Lei Ao Li Jun Li Shuxing Li Lin Chen Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes Microbial Cell Factories Three finger protein E. coli recombinant expression Inclusion body refolding Disulfide bond formation Oxidation refolding |
| title | Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes |
| title_full | Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes |
| title_fullStr | Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes |
| title_full_unstemmed | Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes |
| title_short | Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes |
| title_sort | scalable production of recombinant three finger proteins from inclusion bodies to high quality molecular probes |
| topic | Three finger protein E. coli recombinant expression Inclusion body refolding Disulfide bond formation Oxidation refolding |
| url | https://doi.org/10.1186/s12934-024-02316-1 |
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