Abstract 1583 Rd-Rd fusion system facilitates soluble expression of recombinant human FGF23 in Escherichia coli

Fibroblast growth factor 23 (FGF23) is a non-heparin binding, metabolic FGF which predominantly mediates phosphate homeostasis and vitamin D metabolism in body. FGF23 level alterations in the body cause many diseases in humans, establishing FGF23 as an attractive therapeutic target. Escherichia coli expression system, which provides cost efficient, rapid expression of recombinant proteins with high yield, is often preferred over mammalian systems. However, large scale expression of FGF23 in E. coli in soluble form so far has been challenging due to its tendency to form inclusion bodies. Hence, the main objective of the current project is to express and purify recombinant human FGF23 (rhFGF23) in soluble and biologically active form in E. coli. We developed a soluble tag; His-Rd-Rd which yielded His-Rd-Rd-rhFGF23. The fusion gene encodes for His-Rd-Rd-rhFGF23 was cloned into pET-22b vector and transformed into E. coliRosetta-gami™ 2 (DE3) competent cells (Novagen). His-Rd-Rd-rhFGF23 protein was expressed in Luria-Bertani broth treated with 0.5 mM isopropyl-thio-β-galactopyranoside for 6 hours at 37 °C at 250 r.p.m. The His-Rd-Rd tagged rhFGF23 showed significant increase in solubility compared to untagged rhFGF23. The fusion protein was purified by Ni-sepharose affinity chromatography. Purified fusion protein was then treated with enterokinase to remove His-Rd-Rd solubility tag from rhFGF23. Rd is a low molecular weight, iron containing protein extracted from a hyperthermophile. Rd in its Fe2+-bound form appears in red, giving Rd-Rd-rhFGF23 fusion protein a red color. This provides an additional benefit during purification process since the elution of Rd-Rd-rhFGF23 could be visually detected along the purification column. Therefore, Rd-Rd solubility tag will be a better strategy for recombinant expression and purification of FGF23 to study FGF-mediated metabolic effects and characterizing FGF23 biophysical properties. Further, our novel Rd-Rd solubility tag can be successfully applied to other recombinant proteins prone to form inclusion bodies.