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.