In Silico Identification And Experimental Validation Of Cellular Uptake And Intracellular Labelling Of HaloTag Proteins By A New Cell-permeable P2 Peptide

Research background Cell Penetrateing Peptide(CPP)is a non-viral gene vector capable of carrying proteins and other biological molecules into cells.Objective The purpose of this study is to investigate the penetrating efficiency of a novel cell-penetrating peptide P2,investigate its penetrating property positioning acting as a carrier-mediated self-labeling label protein HaloTag protein,to provide more theoretical support for the further application of P2,a new-type cell penetrateing peptide as a biomolecule transporter in the intracellular delivery vector.Methods(1)According to bio-informational web and structural characteristics of reported CPPs,we designed and synthesised P2-FITC,NCO-FITC and other short peptides with fluorescence labeled.Predict and analyze its physicochemical properties,structural characteristics,penetrating efficiency by bioinformatics.(2)Via in vitro co-incubation of P2 and NCO,the cellular uptake of CPP was detected with fluorescence microscope and quantified with multimode spectrophotometry to analyze intracellular fluorescence of P2 under different conditions.(3)The uptake mechanism of P2 was examined via the influence of temperature and endocytosis inhibitors on peptide internalization.The interaction between P2 peptide and membrane was predicted to further investigate the uptake mechanism of P2 peptide.(4)MTT test was conducted to measure the influence of P2 on cell proliferation,lactic acid dehydrogenase(LDH)releasing assay and erythral lysis experiment were used to evaluate the effect of P2 on cell membrane integrity,which were further studyed if P2 had toxic effects on cells.(5)The p ET15b-HaloTag-P2,p ET15b-HaloTag-Dot1 l,p ET15b-HaloTag prokaryotic expression vectors were constructed.The recombinant HaloTag-P2,HaloTag-Dot1 l,HaloTag were expressied and purification.After cell incubation to add HaloTag ligand TMR to continue incubating,Only TMR acting as blank control,By qualitatively observing the trace location of recombinant HaloTag-P2,HaloTag-Dot1 l,and HaloTag delivery in cells,the delivery efficiency of fusion proteins in cells and the number of cells occupied by positive cells were quantitatively analyzed using Image J software.Results(1)The physicochemical properties,structure and penetrating efficiency of P2 are predicted accurately by bioinformatics.(2)Different concentrations of P2 penetrate can cross cell membrane,distributed in the cell pulp and nuclei,intracellular fluorescence increases with the concentration;P2 membrane penetration efficiency increases over a certain period of short time,intracellular fluorescence increases slightly over time;the penetrating efficiency does not have cell specificity,5% DMSO can significantly improve penetrating efficiency.(3)Low temperature had a slight effect on the penetrating efficiency of P2;the presence of serum,heparin,EIPA,Chlorpromazine inhibited the penetrating efficiency of P2,and the inhibitors Na N3,NH4 Cl,MβCD,wortmannin,and high-permeable sucrose inhibited the penetrating efficiency of P2.(4)P2 had no obvious toxic on the cells.(5)P2 can mediate protein large molecules such as HaloTag protein delivered to cells and distributed mainly in the nucleation of cells.Conclusion(1)P2 is a new type of cell membrane peptide with high penetrating efficiency.(2)experimental results indicated endocytosis was the possible mechanism.(3)P2has no significant cytotoxicity.(4)P2 mediates intracellular delivery of HaloTag proteins.(5)HaloTag-P2 is distributed mainly in the nucleus as a new type of probe,with a small amount located in the cell pulp.