Figure Legends
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.
Figure 2. Analyses of the expression and purification
patterns of cp-hFGF7115-114. (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 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-hFGF7115-114 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-hFGF7115-114.
Figure 3. Spectral profile analyses for the comparison of
structural properties between the wild type and
cp-hFGF7115-114. (A) UV-visible spectra comparison
between the wild type hFGF7 (commercially available) and
cp-hFGF7115-114. (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-hFGF7115-114 was
also analyzed by using the purified proteins (300 µg/mL) under the
specified conditions. Black line, buffer; orange line, rhFGF7; blue
line, cp-hFGF7115-114. 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.
Figure 4. Biological activity of
cp-hFGF7115-114. 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-hFGF7115-114. N.C, negative control. Dose-dependent
ERK1/2 kinase activation in NIH3T3 cells treated with the defined amount
of hFGF7 and cp-FGF7115-114 for 30 min. (C)
Comparisons of cell proliferation activities between hFGF7 and
cp-hFGF7115-114. 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-hFGF7115-114 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.