Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089, USA.
Institute of Clinical Medicine, The First Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, People's Republic of China.
Microb Cell Fact. 2024 Feb 12;23(1):48. doi: 10.1186/s12934-024-02316-1.
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.
三指蛋白是一类具有重要生物医学意义的富含二硫键的蛋白质。可规模化地表达和纯化具有生物活性的三指蛋白颇具难度。
我们提出了一个使用大肠杆菌作为表达宿主的三指蛋白表达、纯化和验证的工作流程。利用该流程，我们成功获得了高度纯化且具有生物活性的重组α-拟蝎毒素、κ-拟蝎毒素、Hannalgesin、Mambalgin-1、α-眼镜蛇毒素、MTα、Slurp1、Pate B 等。在实验室中，数周内可获得数毫克至数百毫克的重组三指蛋白。重组蛋白在结合实验中表现出特异性，其中 6 种蛋白通过 X 射线衍射蛋白质晶体学进行了结晶和结构验证。
我们的流程可在优化条件下对重组三指蛋白进行复性和纯化，并可规模化生产三指蛋白。由于许多三指蛋白具有吸引人的治疗或研究兴趣，并且由于我们获得的重组三指蛋白质量极高，我们的方法为其天然或化学合成的对应物提供了一种有竞争力的替代方案，应有助于相关研究和应用。
