Using GST Pull-down Assay For Screening PAD4Interacting Proteins

GST pull-down assay as a method in study of protein interactions in vitro,it issimple, convenient and easy to operate. But it is difficult to find the interaction withunknown protein, there were rarely reported. Peptidyl arginine deiminase4(PAD4) isa susceptibility gene to Rheumatoid Arthritis (RA). The citrullinated of histone is anecessary condition in formatting the innate immunity-neutrophil extracellular traps(Neutrophil Extracellular Traps, NET) in mammal. PAD4as a inhibitor candownregulate the downstream expression of P53gene which believed to be involvedin tumor occurs, although the specific mechanism is not clear. PAD4is mainlyexpressed in the immune cells, it is the only one of the five peptidyl argininedeiminases has a nuclear localization signal. Our laboratory found PAD4in cellHL-60had a nuclear translocation phenomenon after inducing by TNF-α. PAD4molecular weight becomes larger after translocating to nuclear, suggesting that PAD4could have interacting with other proteins. To clarify its nuclear transport mechanismand find out the interacting protein is necessary to understand gene function in normalphysiological conditions and lesions. In this study we use GST pull-down assay andHL-60cell to have a preliminary exploration.Objective: Reconstruction of GST-PAD4and GST-PAD4-NLS-vectors and thenpurification of GST-PAD4and GST-PAD4-NLS-fusion proteins, using GSTpull-down assay to screen the proteins which have interaction with PAD4whennuclear transferring.Methods: Treate the HL-60cells with TNF-α at different times in order to obvious the PAD4nuclear transfer phenomenon. Construct a GST-PAD4and a GST-PAD4withnuclear localization signal loss (PAD4-NLS-) recombinant plasmid. Both of the twoplasmids are transformated into Escherichia coli BL21to obtain recombinantengineering bacteria. After optimizing the expression conditions to obtain soluble andpurification of the two fusion proteins, purifying the two fusion proteins bySepharose4B affinity chromatography, then the both higher purification fusionproteins are respectively fixed on the column of Sepharose4B, By filling the totalproteins from HL-60cells into the column to enrich the interacting protein with PAD4and PAD4-NLS-and then screen specific proteins finally by10%SDS-PAGE andsilver staining.Results: Nuclear transfer phenomenon of PAD4was observed in HL-60cellafter inducing by TNF-α. TNF-α may have the inducing effect on PAD4nucleartransferring especially after inducing2hrs. By optimizing the soluble expressionconditions of GST-PAD4fusion protein, we got the optimization of soluble expressionconditions of the GST-PAD4fusion protein (induced by0.1mM IPTG over12hrs at16°C when the OD600reach0.4), and then purified of the GST-PAD4andGST-PAD4-NLS-fusion proteins, specific proteins were screened preliminary by10%SDS-PAGE and silver staining which obtained form interacting with PAD4in nucleartransferation after GST pull-down assay. One possible specific protein was screened.Conclution: Observed the phenomenon of PAD4nuclear translocation afterinducing by TNF-α, PAD4molecular weight becomes larger after translocating tonuclear. By optimizing the expression conditions of GST-PAD4fusion protein, weobtained soluble and purification of the both fusion proteins. After using GSTpull-down assay we screened a possible strips which maybe the protein interacttingwith PAD4. These results need to be further analysis.