Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Objective To find small molecules binding specifically to signal transducer and activator of transcription3 (STAT3) based on surface plasmon resonance (SPR) technology and confirm their inhibitory activities to STAT3. Methods The biomolecular interaction analysis T200 system based on SPR technology was used to couple the purified protein STAT3 to CM5 chip under the optimal pH conditions. The compounds with high binding response value were screened out from 50 candidate compounds derived from traditional Chinese medicines and the binding specificity was then confirmed. Biological experiments were performed to confirm the inhibitory effects of the screened compounds on STAT3. The binding pattern of STAT3 and the compound was fitted by molecular docking technique. Results More than 10 candidate molecules exhibited binding activities to STAT3 and kinetics assays revealed that only one candidate molecule, apigenin, showed specific binding. Western-blot analysis exhibited that apigenin inhibited the phosphorylation of STAT3 dose-dependently. Luciferase reporter gene assays demonstrated that apigenin also inhibited IL-6-induced STAT3 transcriptional activity in a dose-dependent manner. Molecular docking results showed that apigenin binds to the SH2 domain of STAT3, and interacts with key residues Glu638, Gln644, Gly656 and Lys658 by hydrogen bonds and with Tyr657 through π-π interactions. Conclusion Apigenin was a direct inhibitor of STAT3.