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 6Ă—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.