1. INTRODUCTION
Fibroblast growth factors (FGFs) are cytokine family proteins comprised of 22 members that function in cell survival, proliferation and differentiation.1 The fibroblast growth factor 7 (FGF7), also known as keratinocyte growth factor 1 (KGF1), is a member of the paracrine-acting FGF family. These proteins are secreted from various types of mesenchymal cells and activate on epithelial cells.2, 3 The activity of human FGF7 (hFGF7) elicits a local signaling response by primarily binding to the high-affinity FGF receptor 2b (FGFR2b) with heparin sulfate/heparin as a cofactor.4 According to previous reports, hFGF7 have functioned well in various human diseases for repairing cells/tissues, diabetic wound healing, cartilage diseases and liver regeneration.2, 5-7 Owing to the increasing demand for the production of hFGF7 with versatile function, the attempts to produce recombinant hFGF7 in various expression hosts including E. colihas been proliferating.8-12
The expression systems in E. coli are widely used for recombinant protein production because of the easy and diverse genetic tools and the availability of high cell density culture techniques. However, many proteins are yet expressed in the soluble fraction due to innate hurdles, such as the limited landscape of protein folding and incorrect pairing of disulfide bonds, leading to unsatisfactory quality and yields of recombinant proteins especially as inclusion bodies.13-15 In this context, hFGF7 is also categorized as a difficult-to-express protein in E. coli . As an existing method to overcome these problems, solubility enhancers, such as Halo tag9 and small peptide with 6×His tag (6HFh8),11 are fused to the recombinant hFGF7. The fusion with 6HFh8 showed high solubility, but the removal of the fusion tag may negatively influence the structure and stability of hFGF7. The codon optimization and/or N-terminal truncation (commercially available Palifermin®) of hFGF7 has also been attempted to enhance the solubility and protein yield in E. coli .12, 16 However, the high yield production of hFGF7 in the soluble fraction is still required especially as an intact protein containing the whole sequence. Therefore, a method for the high-yield production of a recombinant FGF7 without a fusion tag or deletion in E. coli is needed.
Systematic circular permutations (CP) of genes have emerged as a useful tool for conducting studies of polypeptide folding and stability.17 Since the CP-applied protein is simply rearranged without altering the amino acid sequence, the tertiary structure is usually retained.18, 19 Nevertheless, CP can significantly affect stability and activity.19-21Therefore, CP has been adopted by protein engineers to manipulate protein structure and function,22, 23 and has been representatively used as a technique to expand the usefulness of fluorescent proteins.19, 24, 25 According to the current Circular Permutation Database (CPDB), more than 4000 naturally occurring or artificially generated permutation proteins have been identified.26 Nevertheless, there is no report on the generation of CP variants from FGF family proteins.
In this paper, we report the structural and functional characteristics of a CP variant of hFGF7 screened by a typical approach that generated a series of CP mutants by selective polymerase chain reaction (PCR) amplification using the duplicated gene as the template27 and analyzed in terms of expression level and solubility in E. coli . The screened variant cp-hFGF7115-114 showed more stability and productivity than the wild type hFGF7, and was also determined to have comparable activity to the wild type hFGF7.