Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

Xu, Jiang; Lei, Xiao; Li, Ao; Li, Jun; Li, Shuxing; Chen, Lin

doi:10.1186/s12934-024-02316-1

Title: Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

Journal Article · 11 February 2024 · Microbial Cell Factories

DOI: https://doi.org/10.1186/s12934-024-02316-1 · OSTI ID:2471035

Xu, Jiang ^[1]; Lei, Xiao ^[1]; Li, Ao ^[1]; Li, Jun ^[2]; Li, Shuxing ^[1]; Chen, Lin ^[1]

University of Southern California, Los Angeles, CA (United States)
The First Affiliated Hospital of University of South China, Hunan (China)

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. 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. 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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Advanced Light Source (ALS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231; AC02-06CH11357

OSTI ID:: 2471035

Journal Information:: Microbial Cell Factories, Journal Name: Microbial Cell Factories Journal Issue: 1 Vol. 23; ISSN 1475-2859

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biotechnology & Applied Microbiology
Disulfide bond formation
E. coli recombinant expression
Inclusion body refolding
Oxidation refolding
Three finger protein

Title: Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

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