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 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.

Keywords: Disulfide bond formation; E. coli recombinant expression; Inclusion body refolding; Oxidation refolding; Three finger protein.

Fig. 1

Experimental flow chart illustration

Fig. 2

SDS-PAGE analysis of rTFPs at different stages of production. con: control (not induced E. coli cells), pb IPTG induced E. coli cells, I.B. isolated inclusion bodies, purif.: purified final product (in non-reducing SDS-PAGE)

Fig. 3

Structural alignment of the crystal structure of rTFPs and their natural counterpart or most homologous natural counterpart. Yellow: rTFP, Magenta: Reported native (homologous) or synthetic counterpart. a. rec-αBtx-HAP vs αBtx-HAP; b. rec-αCTX vs αCTX; c. rec-κBtx vs κBtx; d. rec-MTα vs MT1; e. rec-Hannalgesin vs αCTX; f. rec-Mambalgin-1 vs Mambalgin-1. RMSD was calculated based on the coordinates of C_α of the aligned structures

Fig. 4

Biochemical characterization of the rTFPs a. Stability of rec-αCTX and rec-Hannalgesin upon prolonged storage at 4 °C, analyzed with non-reducing SDS-PAGE; b. ‘Concentrate to dry’ strategy (right, with black arrow pointing to the monomeric species) efficiently removed multimeric species that were hard to be separated with the mono S column (left, in which the refolded product was not ‘concentrate to dry’, grey arrow); c. Gel filtration analysis of rec-αBtx, rec-mPate B and rec-κBtx. d. Native gel shift assay of various rTFPs with HAP peptide. e. Native gel shift assay of rec-α1ECD with native αCTX, rec-αCTX and rec-Hannalgesin