High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.