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\begin{document}

\title{Construction and characterization of a functional variant hFGF7 with
enhanced properties by circular permutation}



\author[1]{Hye-Ji Choi}%
\author[2]{Hanui Lee}%
\author[1]{Dae-Eun Cheong}%
\author[1]{Su-Kyoung Yoo}%
\author[2]{Dong-Eun Lee}%
\author[1]{Geun-Joong Kim}%
\affil[1]{Chonnam National University College of Natural Science}%
\affil[2]{Korea Atomic Energy Research Institute}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
Human fibroblast growth factor 7 (hFGF7) is a member of the
paracrine-acting FGF family and mediates various reactions such as wound
healing, tissue homeostasis, and liver regeneration. These activities
make it a plausible candidate for pharmaceutical applications as a drug.
However, the low expression level and stability of the recombinant hFGF7
were known to be major hurdles for further applications. Here, we
attempted to improve the expression level and stability of hFGF7 by
changing the order of amino acids through circular permutation (CP),
thereby expecting an alternative fate according to N-end rules. CP-hFGF7
variants were constructed systematically by using putative amino acid
residues in the loop region that avoided the disruption of the
structural integrity especially in the functional motif. Among them,
cp-hFGF7\textsuperscript{115-114} revealed a relatively higher
expression level in the soluble fraction than the wild-type hFGF7 and
was efficiently purified to apparent homogeneity by three consecutive
steps. Approximately, 7 mg of the purified protein was obtained from a 1
L culture and showed a comparable activity to that of the wild type
hFGF7. In addition, the spectral properties related to the apparent
structure of cp-hFGF7\textsuperscript{115-114} were quite similar to
those of the wild type hFGF7. Therefore, CP could be an alternative tool
for the functional expression of hFGF7 in \emph{Escherichia coli}.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Fibroblast growth factors (FGFs) are cytokine family proteins comprised
of 22 members that function in cell survival, proliferation and
differentiation.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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 \emph{E.
coli}has been proliferating.\textsuperscript{8-12}

The expression systems in \emph{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.\textsuperscript{13-15} In this context, hFGF7 is also
categorized as a difficult-to-express protein in \emph{E. coli} . As an
existing method to overcome these problems, solubility enhancers, such
as Halo tag\textsuperscript{9} and small peptide with 6\selectlanguage{ngerman}×His tag
(6HFh8),\textsuperscript{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 \emph{E. coli} .\textsuperscript{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 \emph{E. coli} is needed.

Systematic circular permutations (CP) of genes have emerged as a useful
tool for conducting studies of polypeptide folding and
stability.\textsuperscript{17} Since the CP-applied protein is simply
rearranged without altering the amino acid sequence, the tertiary
structure is usually retained.\textsuperscript{18, 19} Nevertheless, CP
can significantly affect stability and
activity.\textsuperscript{19-21}Therefore, CP has been adopted by
protein engineers to manipulate protein structure and
function,\textsuperscript{22, 23} and has been representatively used as
a technique to expand the usefulness of fluorescent
proteins.\textsuperscript{19, 24, 25} According to the current Circular
Permutation Database (CPDB), more than 4000 naturally occurring or
artificially generated permutation proteins have been
identified.\textsuperscript{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
template\textsuperscript{27} and analyzed in terms of expression level
and solubility in \emph{E. coli} . The screened variant
cp-hFGF7\textsuperscript{115-114} showed more stability and productivity
than the wild type hFGF7, and was also determined to have comparable
activity to the wild type hFGF7.

\textbf{2. MATERIALS AND METHODS}

2.1. Bacterial strains, plasmids, and reagents

\emph{E. coli} XL1-Blue (\selectlanguage{greek}\emph{endA1 gyrA96 {[}nalR{]} thi-1 recA1 relA1
lacglnV44 F`{[}::Tn10 proAB+ lacIqΔ(lacZ)M15{]} hsdR17{[}rK-mK+{]}}\selectlanguage{english} )
was used as a host for the cloning of the recombinant gene, and protein
expression was induced in the host \emph{E. coli} BL21(DE3) (\selectlanguage{greek}\emph{ompT
gal dcm lon hsdSB{[}rB\textsuperscript{--}mB\textsuperscript{--}{]}
λ(DE3 {[}lacI lacUV5-T7p07 ind1 sam7 nin5{]}) {[}malB+{]}K-12{[}λS{]}}\selectlanguage{english}
). pET24a\_hFGF7 was provided by the Korea Institute of Marine Science
and Technology (Busan, Korea) and used as the template for PCR with a
set of primers specially designed for the duplication of the gene
encoding hFGF7 (Table S1). The vector pSCT5\textsuperscript{28} was used
to insert the duplicated hFGF7 gene for the generation of CP variants by
PCR, and the pCold I vector (TaKaRa, Japan) was used to construct a
series of CP hFGF7 variants (cp-hFGF7). n\emph{Pfu} special polymerase
(Enzynomics, Daejeon, Korea) was used for the amplification of the
target gene by PCR. The restriction enzymes used were purchased from New
England Biolabs (Ipswich, MA,~USA\textbf{)} and Takara Bio Inc. (Shiga,
Japan). T4 DNA ligase (Thermo Fisher, MA, USA) or the Infusion HD
Cloning Kit (Clontech, Shiga, Japan) was used for the subcloning of the
target gene into the plasmids according to the supplier's recommended
protocol. Recombinant hFGF7 (indicated as wild type in this work) was
purchased from PeproTech (Cranbury, NJ, USA) and used as a positive
control for the structure and activity analyses of the purified
cp-hFGF7.

2.2. Prediction of CP cleavage sites in hFGF7

CP cleavage sites were predicted by a web-based tool, CPred
().\textsuperscript{26} Among residues with a probability score of
greater than 0.8, the candidate positions for creating new termini were
rationally selected from surface loops that predicted also without
significant loss of structural traits by the secondary structure
prediction using PSIPRED.\textsuperscript{29}

2.3. Construction and analyses of the expression patterns of circularly
permuted hFGF7 variants

For the construction of diverse CP variants, the gene encoding hFGF7
without an innate signal sequence (31 amino acids) was primarily
duplicated by artificial fusion using PCR as follows. The DNA fragment
encoding 163 amino acid residues of hFGF7 was amplified by PCR using
pET24a\_hFGF7 as the template and two sets of primers
(pSCT5\_hFGF7-Infu-F and hFGF7-Infu-R, hFGF7(\selectlanguage{ngerman}×2)-Infu-F and
pSCT5\_hFGF7(×2)-Infu-R) under the typical conditions. Then, the
resulting DNA fragment was cloned into a linearized pSCT5 vector to
prepare the construct pSCT5\_hFGF7(×2) containing the duplicate gene
(Figure 1A). Using the pSCT5\_hFGF7(×2) vector as the template, CP
variants were amplified by PCR using nine sets of primers
(pSCold\_CP1-hFGF7-F/R \textasciitilde{} pSCold\_CP9-hFGF7-F/R), and
subcloned into the same restriction enzyme site (\emph{Spe} I
and\emph{Hin} dIII) to prepare pSCold\_cp-hFGF7 constructs expressing
each of the nine CP variants (Figure 1B, C). Prior to the subcloning of
cp-hFGF7, the removal of TEE-6×His-fXa cleavage sequence from the
expression vector pCold I and incorporation of the\emph{Spe}
I-recognizing sequence ACTAGT (between 5'UTR and start codon) were
simultaneously carried out by PCR using a pair of primer
(pSCold\_Vec-Infu-R and pSCold\_Vec-Infu-F).\textsuperscript{28} The
resulting vector pSCold could express cp-hFGF7 without any additional
amino acids at N-terminal region. All primer sequences used in this
study are provided in Table S1.

To analyze the expression level and solubility of the cp-hFGF7 variants,
each of the recombinant plasmid pSCold\_cp-hFGF7 variants (CP1--CP9) was
transformed into\emph{E. coli} BL21(DE3) cells. Subsequently, a single
colony was inoculated into 3.5 mL of LB medium containing ampicillin
(100 \selectlanguage{greek}µ\selectlanguage{english}g/mL) and cultured at 37 under constant shaking (200 rpm). When
the absorbance (OD\textsubscript{600}) of the culture reached 2.0, an
aliquot of culture broth was reseeded (2\%, v/v) into the same LB
medium. The resulting cells were cultured to an OD\textsubscript{600} of
0.6 and treated with isopropyl-\selectlanguage{greek}β-\selectlanguage{english}D-thiogalactoside (IPTG, 0.2 mM) to
induce protein expression at 16 and 200 rpm for 36 h. The induced cells
were then harvested by centrifugation at 12,000 \selectlanguage{ngerman}× g for 10 min and
resuspended in 10 mM sodium phosphate buffer (pH 6.5), then disrupted by
irradiation with ultrasonic waves three times for 2 seconds. The
resulting cell lysate (total fraction, T) was centrifuged at 4\selectlanguage{english} and
12,000 x g for 25 min to obtain a supernatant (soluble fraction, S) from
which insoluble aggregates had been removed. Both total and soluble
fractions were loaded onto a Tricine-SDS-PAGE (10\%) gel, and the
expression level and solubility were analyzed under the same conditions
previously reported.\textsuperscript{30} The resulting gels were also
subjected to western blot analyses as follows. The transfer of resolved
proteins from gels onto nitrocellulose membrane (GenDEPOT, Texas, USA)
was conducted using a Power Blotter-Semi-dry transfer system
(Thermo-Fisher Scientific, MA, USA). The membrane was then blocked using
5\% skim milk for 1 h at room temperature (RT), followed by incubating
overnight at 4degC with anti-human FGF7 monoclonal antibody (1:5000,
Abcam, US). Subsequently, the membrane was incubated with horseradish
peroxidase-linked goat anti-mouse immunoglobulin G (1:5000, Enzo Life
Sciences, US) at RT for 1 h. The proteins on the membrane were
visualized with ECL detection kit system (Bio-Rad, US).

2.4. Purification of cp-hFGF7

The recombinant plasmid, pSCold\_cp-hFGF7\textsuperscript{115-114}, was
transformed into \emph{E. coli} BL21(DE3) cells, and cultured at 37\selectlanguage{english}
until the cell density (OD\textsubscript{600}) reached 2.0 in 4 mL of LB
medium containing ampicillin (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL). Then, 1 mL of the cultured
cells was reseeded into fresh LB medium (100 mL) and cultured to an
OD\textsubscript{600} of 0.6--0.8, then induced with 0.2 mM IPTG at 16
and 200 rpm for 36 h. The induced cells were harvested by
centrifugation, resuspended in 20 mM sodium phosphate (pH 6.5) buffer,
lysed by sonication, and centrifuged at 4 and 10,000 \selectlanguage{ngerman}× g for 60 min to
remove the cell debris. Using the resulting supernatant,
cp-hFGF7\textsuperscript{115-114} was purified via a successive step
consisting of heparin affinity, cation exchange, and size exclusion
chromatography (SEC). Considering the functional structure for cofactor
binding, the heparin HP column (1 mL, GE Healthcare, IL, USA) was
selected for the primary step. The supernatant was then loaded onto a
heparin column equilibrated with buffer A (20 mM sodium phosphate, pH
6.5). After binding, the column was thoroughly washed with the same
buffer containing 0.2 M NaCl. Thereafter, a linear gradient was induced
with 30 column volumes (CV) of buffer A and B (20 mM sodium phosphate, 1
M NaCl, pH 6.5). Fractions containing
cp-hFGF7\textsuperscript{115-114}were collected and diluted 2-fold with
a buffer (20 mM sodium phosphate, pH 7.3). Next, the eluted fractions
from the heparin column were loaded onto a HiTrap SP HP column (5 mL, GE
Hewlett, IL, USA) equilibrated with the same buffer. After complete
washing with the same buffer, the bound proteins were eluted with 20 CV
of the linear salt (NaCl) gradient buffer from 0 to 1.0 M. The final
step of the purification was conducted using a Superdex 200 increase
10/300 GL column (GE Healthcare, Chicago, USA) with 20 mM sodium
phosphate buffer (pH 7.3) containing 0.4 M NaCl. The purity and yield of
the protein in the eluted fraction were determine by 10\%
Tricine-SDS-PAGE and western blot.

2.5. Spectroscopic property analyses of cp-hFGF7

To analyze the structural property of cp-hFGF7\textsuperscript{115-114},
UV-vis absorption scanning was performed by using a buffer (20 mM sodium
phosphate, 0.4 M NaCl, pH 7.3) under the specified conditions. The
protein solution (100 \selectlanguage{greek}μ\selectlanguage{english}L) was placed into a 1.0 cm quartz cuvette and
the absorbance spectrum was measured by changing the wavelength from 260
to 600 nm at 5 nm intervals.\textsuperscript{31} Fluorescence emission
scanning was also performed by using the same concentration (150 \selectlanguage{greek}µ\selectlanguage{english}g/mL)
of proteins. The change in fluorescence wavelength emitted from 280 to
500 nm was measured at 3 nm intervals by an excitation wavelength of 250
nm.\textsuperscript{32}

The secondary structure analysis of cp-hFGF7\textsuperscript{115-114}was
performed using a circular dichroism (CD) spectropolarimeter (Model
J-1500, Jasco, Tokyo, Japan). Prior to CD measurement, the purified
cp-hFGF7\textsuperscript{115-114} from the size exclusion column was
completely desalted in a 10 mM sodium phosphate (pH 7.3) buffer by using
a PD-10 (GE Healthcare, IL, USA) column. The far-UV CD spectra of
cp-hFGF7\textsuperscript{115-114} (300 \selectlanguage{greek}µ\selectlanguage{english}g/mL) was recorded from 190 to
260 nm using a 0.1 cm path length cell at room temperature (25). Each
spectrum was obtained three times at a scan rate of 100 nm/min, and then
corrected by subtracting the spectral contribution of the buffer. The
commercially available hFGF7 was used as a control for all spectral
experiments.

2.6. Biological activity analyses of cp-hFGF7

\textbf{Extracellular signal-regulated kinase (ERK) phosphorylation
assay} . The~embryonic mouse fibroblast~cell line~NIH3T3 was obtained
from the lab of Professor Tae-Hoon Lee at Chonnam National University
and routinely maintained in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 10\% bovine calf serum (BCS) and
penicillin-streptomycin (100 U/mL and 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL, respectively) in an
incubator (5\% CO\textsubscript{2}) at 37. For immunoblotting,
2\selectlanguage{ngerman}×10\textsuperscript{5} NIH3T3 cells were seeded onto 6-well plates in
the same medium and cultured overnight. The cells were then
serum-starved for 24 h prior to treatment with
cp-hFGF7\textsuperscript{115-114}. After time- and dose-dependent
treatment with cp-hFGF7\textsuperscript{115-114}, the treated cells were
harvested and lysed using RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM
NaCl, 1\% NP-40, 1\% sodium deoxycholate, and 0.1\% SDS) containing
protease and phosphatase inhibitor cocktails. The lysed cells were
centrifuged at 4\selectlanguage{english} and 12,000 x g for 20 min to obtain the supernatant.
The protein concentration in the supernatant was measured using the BCA
protein assay kit (Thermo Fisher, MA, USA). Then, the same amount of
protein from each cell was separated by 10\% Tricine-SDS-PAGE and
transferred to a nitrocellulose membrane. The membrane was blocked in
TBS-Tween 20 (0.1\%) containing 5\% skim milk at room temperature.
Specific proteins on the membranes were detected by probing with
specific primary antibodies, anti-phospho-specific ERK-1/2
(Thr202/Tyr204) and anti-ERK-1/2 antibodies from Cell Signaling
Technology Inc. (Beverly, MA, USA) and the \selectlanguage{greek}α-\selectlanguage{english}tubulin antibody from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA, USA), followed by incubation
with the secondary antibodies conjugated to HRP (Enzo Life Sciences, MI,
USA). The resulting specific binding was visualized by the ChemiDoc
image analyzer (Bio-Rad, CA, USA) using an ECL chemiluminescence
substrate (Bio-Rad, CA, USA).

\textbf{Cytotoxicity and cell proliferation assay} . Cytotoxicity
analysis was carried out by live cell counting using NIH3T3 cells and an
assay kit (Abcam, Cambridge, UK). Cells grown in the same medium
described above were inoculated into 24-well plates and incubated for
24--36 h in a humidified incubator (37) containing 5\%
CO\textsubscript{2}. When the cell confluence reached 70--80\%, a new
medium containing commercial hFGF7 and
cp-hFGF7\textsuperscript{115-114}was added. After incubation for 24 to
72 h, 10 \selectlanguage{greek}μ\selectlanguage{english}L of WST-8 dye was added and an additional incubation was
performed for 3 h. Afterward, the degree of color change was measured at
450 nm using a spectral microplate reader (SpectraMax ABS, Molecular
devices, CA, USA). The results were expressed as a percentage of the
control where the absorbance value of the untreated cells was normalized
to 100\%. After the cell proliferation proceeded in the same manner, the
degree of color change of the WST-8 dye was measured at 460 nm. All
assays were performed in triplicate.

\textbf{Scratch wound healing assay} . NIH3T3 cells used in the wound
healing assay were inoculated into 24-well plates and cultured until
90\% to 100\% cell confluency was reached. A scratch wound was
introduced with a 10 \selectlanguage{greek}μ\selectlanguage{english}L pipette tip. After washing with serum-free DMEM
medium for cell debris removal after scratch formation, the cells were
treated with rhFGF7 and cp-hFGF7\textsuperscript{115-114} and incubated
for 72 h. During the incubation period, sutures for wound closing were
monitored and imaged with an optical microscope (Eclipse TE2000-E,
Nikon, Tokyo, Japan).\textsuperscript{33}

\textbf{3. RESULTS AND DISCUSSION}

\textbf{3.1. Construction and expression analyses of circularly permuted
variants cp-hFGF7s}

Conventional approaches that have attempted to produce the recombinant
hFGF7 in \emph{E. coli} have limitations in improving soluble expression
and stability.\textsuperscript{8, 9, 11} As an alternative, we attempted
to use a typical technique for inducing changes in the N- and C-terminal
ends through the generation of new termini by using CP. CP, a phenomenon
of rearrangement of such protein structures, can alter functional
properties such as solubility and/or stability by generating new
terminal regions from interior residues. Since a variant of the cytokine
interleukin 4 had been successfully reported,\textsuperscript{34} the CP
technique has been presumed to be applicable for producing variants with
novel properties in small single-domain proteins.

Practically, plausible CP sites had been selected through the
combination of prediction results from two web-based tools
(CPred\textsuperscript{26} and PSIPRED\textsuperscript{29}) because a
deposited 3D structure (PDB: 1QQK) of hFGF7 was generated from a variant
with N-terminal deletion and thus had different sequence from the wild
type hFGF7 (Accession No. P21781) (Figure S1). Two high-scoring sites in
a long loop (K100 to N107) and heparin binding region (K149 to
K153)\textsuperscript{35, 36} were arbitrary excluded to retain
structural and functional integrity (Table S2). Using the selected
sequences, specific PCR was performed using the tandemly duplicated
hFGF7 gene as a template and a set of primers spanning the permutable
loop residues (Figure 2A). The resulting DNAs were subcloned into a
redesigned vector pSCold that removed the TEE-6\selectlanguage{ngerman}×His-fXa cleavage
sequence from the original pCold I vector, enabling direct comparison of
the expression level and solubility of cp-hFGF7 to those of the wild
type hFGF7 produced without any tag at the N-terminus. As shown in
Figure 2B, when the protein expression was induced with 0.2 mM IPTG at
16\selectlanguage{english} for 36 h, the expression pattern of each clone harboring a
pSCold\_cp-hFGF7 variant was quite different from each other. Five CP
variants were expressed distinctly and three variants were hardly
detected in the soluble and/or insoluble fractions. Changes in the
cultivation conditions under different media and temperatures did not
significantly improve the expression properties. Intriguingly, the CP
variant cp-hFGF7\textsuperscript{115-114} revealed an enhanced
expression level and solubility when compared with those of the wild
type hFGF7, thus it was selected to be a candidate for further analyses.

\textbf{3.2. Purification of cp-hFGF7\textsuperscript{115-114}}

The variant cp-hFGF7\textsuperscript{115-114} with an enhanced
expression level and solubility was purified to apparent homogeneity
using the three successive steps consisting of a heparin affinity,
cation-exchange, and size exclusion chromatography. To test whether the
innate binding ability of hFGF7 to heparin was preserved in the purified
variant, an affinity chromatography was primarily performed under the
same conditions used for the wild type hFGF7.\textsuperscript{37} As
shown in Figure S2A, cp-hFGF7\textsuperscript{115-114} was eluted in the
fractions corresponding to the concentrations of 0.75 to 0.85 M NaCl
after binding to the heparin column (Figure S2B). This result was
comparable to those of the wild type hFGF7 that eluted with 0.5 to 0.6 M
NaCl,\textsuperscript{37} and thus provide a possibility that
cp-hFGF7\textsuperscript{115-114} was able to bind heparin more tightly
by a subtle change of the heparin binding site\textsuperscript{38} via
CP. The structurally solid conformation of
cp-hFGF7\textsuperscript{115-114} was also partly proven by reproducible
binding patterns to the heparin column. In the subsequent purification
step using a cation-exchange column,
cp-hFGF7\textsuperscript{115-114}was reproducibly eluted from the column
with a salt concentration of approximately 0.7 M NaCl (Figure S2C). To
remove the minor impurities remaining in the fractions from the
ion-exchange column, we finally performed SEC and obtained a symmetrical
peak corresponding to the target protein
cp-hFGF7\textsuperscript{115-114} (Figure S2D). The retention time of
the eluted peak was further compared to those of the protein molecular
weight markers and calculated to be a monomeric protein with a molecular
mass of 19 kDa. This value was perfectly consistent with that of the
wild type protein. As shown in Figure 2C, the purified protein was to
apparent homogeneity (\textgreater{}95\%) in Tricine SDS-PAGE and was
reproducibly obtained via the purification procedures. The final yield
of the purified cp-hFGF7\textsuperscript{115-114} from the flask culture
of the recombinant BL21(DE3) was approximately 7 mg/L. This was 2.5-fold
higher than that of the previously reported result using \emph{E. coli}
as a host.\textsuperscript{8}

Prior to the structural and functional characterizations, the storage
stability of cp-hFGF7\textsuperscript{115-114} was compared to the
commercially available wild type rhFGF7 under the same storage
conditions at 4\selectlanguage{english} for 35 days. As shown in Figure S3, the SDS-PAGE
results revealed that cp-hFGF7\textsuperscript{115-114} had comparable
or greater stability than the wild-type hFGF7. The wild type hFGF7 was
innately unstable and thus susceptible to proteolytic
attack\emph{in-vivo} and/or \emph{in-vitro} even after purification to
apparent homogeneity. Taken together, the screened variant
FGF7\textsuperscript{115-114} could have promising properties in terms
of the expression level and stability, and thus was subjected to further
analyses in structure and activity for comparison to the wild type
protein.

\textbf{3.3. Structural properties ofcp-hFGF7\textsuperscript{115-114}}

To analyze the effect of CP on the structure of FGF with a single
domain, UV-vis absorption, fluorescence spectroscopy, and the circular
dichroism spectra of cp-hFGF7\textsuperscript{115-114} were used and the
results were compared to that of the wild type protein when necessary.
These spectral properties were closely linked with the apparent
structure (secondary and/or tertiary structure) of proteins. UV-vis
absorption and fluorescence emission wavelengths are known as simple
methods capable of confirming structural changes by monitoring the
overall spectrum using photometric and fluorometric spectral
scanning.\textsuperscript{39} As shown in Figure 3A, there is also no
distinct difference between cp-hFGF7\textsuperscript{115-114} and hFGF7
in the adsorption spectra although the spectra were not overlapped due
to the different concentration of proteins. Additionally, the
fluorescence spectrum of cp-hFGF7\textsuperscript{115-114}perfectly
overlapped with that of the wild type hFGF7 when excited at 250 nm and
then emission scanned from 280 to 500 nm under the same protein
concentration (200 \selectlanguage{greek}μ\selectlanguage{english}g/mL, Figure 3B). These results partly suggested
that the difference in the apparent structure between the two proteins
were negligible. We further analyzed the secondary structure of the
purified cp-hFGF7\textsuperscript{115-114} by using far-UV CD and
compared it to the previously reported result. As shown in Figure 3C,
cp-hFGF7\textsuperscript{115-114} showed typical absorption bands at 193
to 223 nm, which are characteristic bands of the secondary structures of
proteins. The contents of the calculated secondary structure based on
the CD spectra are shown in Table S3. When compared to those of the
reported results,\textsuperscript{8} there is a slight difference in the
contents of the secondary structure between the reported hFGF7 and
cp-hFGF7\textsuperscript{115-114} structures. These differences may be
due to the remaining 6\selectlanguage{ngerman}×His tag in the reported hFGF7 after affinity
purification using Ni-Sepharose column.\textsuperscript{8} There is also
a possibility that CP marginally affected the secondary structure.
Additionally, the equipment and buffer composition used between the two
experiments were not the same.

\textbf{3.4. ERK1/2 kinase activation activity of
cp-hFGF7\textsuperscript{115-114}}

hFGF7 can trigger Ras-Raf-ERK1/2 MARK pathway signaling by direct
interaction with the FGFR2b receptor, thereby further stimulating
reactions such as epithelial cell proliferation and
migration.\textsuperscript{40} Based on the previously reported works
for mouse and human cell-based functional analysis of
hFGF7,\textsuperscript{41, 42} we also evaluated whether the MARK
signaling pathway was activated by recombinant
cp-hFGF7\textsuperscript{115-114} using a mouse embryonic fibroblast
NIH3T3 cell. NIH3T3 cells were treated with the purified
cp-hFGF7\textsuperscript{115-114}, and ERK1/2 phosphorylation in these
cells was evaluated in a time- and dose-dependent
manner.\textsuperscript{43} As shown in Figure 4A,
cp-hFGF7\textsuperscript{115-114} (50 ng/mL) induced clear
phosphorylation of ERK1/2, and its effect decreased in a time-dependent
manner. In addition, cp-hFGF7\textsuperscript{115-114} also activated
ERK1/2 phosphorylation in a dose-dependent manner that was similar to
the commercially available wild type protein (Figure 4B). These results
provided strong evidence that the CP did not profoundly change the
biological activity of cp-hFGF7\textsuperscript{115-114} and thus
retained a capability for phosphorylation of ERK1/2 in experimental cell
lines.

\textbf{3.5. Cell proliferation activity of
cp-hFGF7\textsuperscript{115-114}}

The biological function of hFGF7 can also be proven by the controlling
process for repairing epithelial cells \emph{in-vitro} ; therefore, we
further tested whether cp-hFGF7\textsuperscript{115-114} could induce
the proliferation of NIH3T3 cells. A cell proliferation assay for time
course analyses was conducted at 1, 10, and 50 ng/mL
cp-hFGF7\textsuperscript{115-114} concentrations by using purified
proteins. As shown in Figure 4C,
cp-hFGF7\textsuperscript{115-114}retained a cell proliferation activity
similar to that of the wild type hFGF7 under the same concentrations and
incubation times. We also measured the cellular toxicity of
cp-hFGF7\textsuperscript{115-114} by using NIH3T3 cells. To do so, the
cells were treated with cp-hFGF7\textsuperscript{115-114} for 24 and 72
h at concentrations of 0.5, 1, 10, 50, and 100 ng/mL. The commercially
available hFGF7 was also used as the control. As a result, a survival
rate of 98 to 100\% was shown in the case of the purified
cp-hFGF7\textsuperscript{115-114} when the cells were treated for 24 h
at a concentration of 50 ng/mL. At the 72 h culture time point, the cell
viability did not decrease to 95\% or less even with the purified
protein at a concentration of 100 ng/mL (Figure S4). These results
confirmed that the purified cp-hFGF7\textsuperscript{115-114} did not
cause more cytotoxicity than the wild type, and thus we were unable to
observe a distinct difference between the two proteins.

\textbf{3.6. Migration and would healing assay of
cp-hFGF7\textsuperscript{115-114}}

\emph{In-vitro} scratch wound healing analysis was performed using
NIH3T3 cells to evaluate the association between ERK activation and cell
proliferation in FGF7-stimulated cells. As shown in Figure 4D, untreated
NIH3T3 cells did not show migration after 72 h, whereas cells treated
with cp-hFGF7\textsuperscript{115-114} and hFGF7 (50 ng/mL) revealed
cell proliferation and wound repair by migration after 48 h. In this
result, cp-hFGF7\textsuperscript{115-114} displayed comparable or better
cell proliferation and wound healing ability than that of the wild type
hFGF7. Additionally, it was found that cell proliferation was difficult
to induce as that of the wild type hFGF7 using a low concentration of
cp-hFGF7 (1 ng/mL). These results confirmed that
cp-hFGF7\textsuperscript{115-114} retained the same or higher activity
than the commercially available rhFGF7.

Since the potential activities for cell proliferation and migration were
discovered, FGF7 has been considered a practical drug candidate. In
fact, although it is not an intact form, the variant Palifermin with a
24-amino acid deletion from the N-terminal region is commercially
available as a drug for the treatment of oral
mucositis.\textsuperscript{38} Therefore, the wild type hFGF7 and its
derivatives with suitable activity and stability would have potential
for practical applications.

\textbf{4. CONCLUSION}

We herein developed the circularly permuted variant
cp-hFGF7\textsuperscript{115-114} without any deletion of the wild type
hFGF7. This protein proved to be more soluble and stabile than the wild
type, and had comparable or greater biological activity than the wild
type protein. Taken together, although more sophisticated analyses and
preclinical trials are needed for practical application, we expect that
the variant cp-hFGF7\textsuperscript{115-114} could be a potential
alternative drug candidate for related symptoms. In addition, the CP
technique was also broadly applicable for the generation of functionally
soluble variants of the FGF family proteins.

\textbf{CRediT authorship contribution statement}

\textbf{Hye-Ji Choi:} ~Investigation, Visualization, Writing -- original
draft.~\textbf{Hanui Lee:} ~Investigation, Formal analysis.
\textbf{Dae-Eun Cheong:} ~Methodology, Validation, Writing -- original
draft.~~\textbf{Su-Kyoung Yoo} : Visualization, Formal
analysis.\textbf{Dong-Eun Lee:} ~Methodology,
Resources.~\textbf{Geun-Joong Kim:} ~Conceptualization, Methodology,
Supervision, Writing -- original draft, Writing -- review \& editing,
Project administration.

\textbf{Declaration of Competing Interest}

Geun-Joong Kim, Hye-Ji Choi, Hanui Lee, Dae-Eun Cheong and Su-Kyoung Yoo
declare that they have conflict of interest as the method for preparing
recombinant FGF7 through circular permutation has been filed as patents
(Korean Patent Application No. 10-2022-0051241, United States of America
Patent Application No. 18/061,836). Dong-Eun Lee declare no competing
interests.

\textbf{Data availability statement}

All data generated or analyzed during this study were included in this
published article (and its supplementary information files).

\textbf{Acknowledgement}

This work was supported by a grant from the Marine Biotechnology Program
(Grant No. 20170305) funded by the Ministry of Oceans and Fisheries,
Korea. This work was also partly supported by the Commercializations
Promotion Agency for R\&D Outcomes (COMPA) grant (No. 2021C300) funded
by the Korea government (MSIT) and Basic Science Research programs
(Grant No. 2021R1A2C1006734) of the National Research Foundation (NRF)
of Korea funded by the Ministry of Education, Science and Technology of
Korea (MEST).

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\textbf{Figure Legends}

\textbf{Figure 1. Schematic diagram for the construction and cloning of
cp-hFGF7 variants.} (A) Genetic organization of the vector including a
template polynucleotide for PCR amplification of the circular
permutation (CP) fibroblast growth factor (FGF7) variants, in which the
template was prepared by gene duplication. (B) After a tandem gene was
constructed without a linker, circular permutants with new start (n) and
stop (n-1) residues of hFGF7 were generated using PCR, then subcloned
into the expression vector pSCold. The residues of cp-hFGF7 are numbered
according to hFGF7 sequence without the signal peptide. (C) Genetic
organization of the expression vector containing a cp-hFGF7 variant for
the inducible expression under a low temperature.

\textbf{Figure 2.} \textbf{Analyses of the expression and purification
patterns of cp-hFGF7\textsuperscript{115-114}.} (A) To visualize the
selection results of circular permutation (CP) sites from the two
web-based tools, CP site-containing loops are marked as yellow lines in
the deposited tertiary structure of hFGF7 in the data bank (PDB ID:
1QQK). (B) The expression patterns of the constructed variants of
cp-hFGF7 in \emph{E. coli} BL21 (DE3) were analyzed by Tricine-SDS-PAGE
under denaturing conditions. Western blots were also analyzed using the
same PAGE gels. (C) The eluted fractions contacting
cp-hFGF7\textsuperscript{115-114} from each chromatography were analyzed
by Tricine-SDS-PAGE and western blot under the same conditions described
in the Materials and Methods section. M, molecular weight markers; T,
total fraction; S, soluble fraction; FT, flow through. Arrows indicate
the protein band corresponding to cp-hFGF7\textsuperscript{115-114}.

\textbf{Figure 3. Spectral profile analyses for the comparison of
structural properties between the wild type and
cp-hFGF7\textsuperscript{115-114}.} (A) UV-visible spectra comparison
between the wild type hFGF7 (commercially available) and
cp-hFGF7\textsuperscript{115-114}. (B) Fluorescence spectra of both
proteins were scanned from 280 to 500 nm by excitation at 250 nm. (C)
The circular dichroism spectrum of cp-hFGF7\textsuperscript{115-114} was
also analyzed by using the purified proteins (300 \selectlanguage{greek}µ\selectlanguage{english}g/mL) under the
specified conditions. Black line, buffer; orange line, rhFGF7; blue
line, cp-hFGF7\textsuperscript{115-114}. All experiments were conducted
at least three times according to the described procedure in the
Materials and Methods section and their average values were plotted in
these figures.

\textbf{Figure 4. Biological activity of
cp-hFGF7\textsuperscript{115-114}.} Dose- (A) and time-dependent (B)
extracellular signal-regulated kinase (ERK) phosphorylation activity
analyses in NIH3T3 cells. A time course of ERK1/2 phosphorylation is
shown in NIH3T3 cells treated with 50 ng/mL of wild type hFGF7 and
cp-hFGF7\textsuperscript{115-114}. N.C, negative control. Dose-dependent
ERK1/2 kinase activation in NIH3T3 cells treated with the defined amount
of hFGF7 and cp-FGF7\textsuperscript{115-114} for 30 min. (C)
Comparisons of cell proliferation activities between hFGF7 and
cp-hFGF7\textsuperscript{115-114}. The same amounts (1, 10, and 50
ng/mL) of both proteins were treated under the identical conditions and
their activities were monitored over time. (D) Comparison of scratch
wound healing activity between hFGF7 and
cp-hFGF7\textsuperscript{115-114} in NIH3T3 cells. After scratching, the
cells were treated with a defined amount of both proteins under the same
conditions and their effects on would healing were monitored for 72 h.
All experiments were conducted at least three times according to the
described procedure in the methods section. The commercially available
hFGF7 was designated as the wild type and used as the positive control.\selectlanguage{english}
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