Expression And Purification Of Bioactive Recombinant Human Fibroblast Growth Factor18in Escherichia Coli And Its Effects On Hair Growth

Using fusion tags, expression of recombinant human fibroblast growth factor18(rhFGF18)in mammalian cells and Escherichia coli (E.coli) has been extensively used for fundamentalresearch and clinical applications, including chondrogenesis and osteogenesis, hair growth, andneuroprotection. However, high-level rhFGF18expression is difficult and the products are oftennot homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lowerbioactivity, and the removal of the fused tag is expensive. To overcome the limitations offusion-tagged expression of protein and to prepare soluble highly-bioactive rhFGF18, we havedeveloped a rapid and efficient expression strategy.Optimized hFGF18gene was amplified by PCR and cloned into pET22b(+) and pET3cvectors, then transformed into E.coli strains Origima (DE3) and BL21(DE3)PlysS. The bestcombination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18was screened for high-level expressed rhFGF18. Under optimal conditions, the expression levelof rhFGF18was30%, after treatment with1mM isopropyl-thio-β-galactopyranoside (IPTG) for10h at25°C. According to the isoelectric point of target protein, the target protein was purifiedby CM Sepharose FF and heparin affinity chromatography, and western blotting analysis with aspecific anti-human FGF18antibody showed a specific reaction with the target protein. Thepurity of rhFGF18was shown by HPLC to be higher than95%. In vitro MTT assaysdemonstrated the purified rhFGF18could stimulate significant proliferation of NIH3T3cells.When rhFGF18was administered subcutaneously to C57BL/6mice in a uniform telogen state,anagen hair growth was conmpared between the rhFGF18-and PBS-administered mice. Theonly certainty is that the effects of rhFGF18on hair follicle growth could be applied in furtherresearch and therapeutic applications. Like other FGFs, sensitivity to temperature andproteolytic enzymes significantly impedes its clinical applications. Fortunately, we establish anovel solid-phase strategy for rhFGF18PEGylation.In the study, the solid-phase modification mainly reacted in the Heparin-Sepharose column,firstly rhFGF18proteins were loaded onto the column and then the reaction liquid contain20kDa mPEG-butyraldehyde as mobile phase was circulated in the column at low flow rate forsome time. Considering the difference of heparin affinity between rhFGF18and PEG-rhFGF18,we directly did the purification in the Heparin-Sepharose column, at the end the reactioncomplex was completely eluted by applying different salt liquid. Under optimal conditions (pH6.5， the matio between PEG and protein was8:1, reaction for12h), the yield ofmono-PEGylated rhFGF18was47.8%. MALDI-TOF-MS showed the molecular weight ofmono-PEG-rhFGF18was40.6kDa, strongly indicated one molecule PEG attachs to theN-terminus of one molecule rhFGF18. In addition, the NIH3T3cells showed that thesolid-phase PEGylated rhFGF18has an unchanged cell proliferation activity. Circular dichroism(CD) spectroscopy analysis revealed that compared to native rhFGF18, the secondary structureof PEGylated rhFGF18were no significant change. Furthermore, the results of the thermalstability and the potent resistance to proteolysis experiments of both wild-type and PEGylatedrhFGF-18indicated the significantly increased thermal tolerance and resistance to proteolysis ofPEGylated rhFGF18. In this research, we acquired the method of solid-phase site-specificPEGylation of rhFGF18process, the modified products not only retained the bioactivities, butalso improved bio-and thermal-stability. In conclusion, this may be a better method ofproducing rhFGF18to meet the increasing demand in its pharmacological application.