3.2. Purification of cp-hFGF7115-114
The variant cp-hFGF7115-114 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 hFGF7.37 As
shown in Figure S2A, cp-hFGF7115-114 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,37 and thus provide a possibility that
cp-hFGF7115-114 was able to bind heparin more tightly
by a subtle change of the heparin binding site38 via
CP. The structurally solid conformation of
cp-hFGF7115-114 was also partly proven by reproducible
binding patterns to the heparin column. In the subsequent purification
step using a cation-exchange column, cp-hFGF7115-114was 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-hFGF7115-114 (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 (>95%) in
Tricine SDS-PAGE and was reproducibly obtained via the purification
procedures. The final yield of the purified
cp-hFGF7115-114 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 E. coli as a
host.8
Prior to the structural and functional characterizations, the storage
stability of cp-hFGF7115-114 was compared to the
commercially available wild type rhFGF7 under the same storage
conditions at 4℃ for 35 days. As shown in Figure S3, the SDS-PAGE
results revealed that cp-hFGF7115-114 had comparable
or greater stability than the wild-type hFGF7. The wild type hFGF7 was
innately unstable and thus susceptible to proteolytic attackin-vivo and/or in-vitro even after purification to
apparent homogeneity. Taken together, the screened variant
FGF7115-114 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.
3.3. Structural properties ofcp-hFGF7115-114
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-hFGF7115-114 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.39 As shown in Figure 3A, there is also no
distinct difference between cp-hFGF7115-114 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-hFGF7115-114perfectly 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 μg/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-hFGF7115-114 by using far-UV CD and
compared it to the previously reported result. As shown in Figure 3C,
cp-hFGF7115-114 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,8 there is a slight difference in the
contents of the secondary structure between the reported hFGF7 and
cp-hFGF7115-114 structures. These differences may be
due to the remaining 6×His tag in the reported hFGF7 after affinity
purification using Ni-Sepharose column.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.