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.