Effects Of Macromolecular Crowded Environment In Goblet Cells Simulated In Vitor On Folding And Aggregation Of Different Glycosylated MUC5AC

Objective: To investigate the different effects of macromolecular crowding on the folding and aggregation of MUC5 AC with different levels of glycosylation during refolding.Methods: Firstly,the eukaryotic expression vector containing the target gene fragment was transfected into HEK293 cells to induce the expression of recombinant MUC5 AC.The protein was purified by nickel column.Then,the purified MUC5 AC was treated with PNGase F and the results of glycosylation were analyzed by SDS-PAGE.Macromolecular crowded environment was simulated by BSA,PEG2000,Ficoll70 and Dextran70.The concentrations were set as 50g/L,100 g/L and 200 g/L.Both of deglycosylated protein(d-MUC5AC)and glycosylated natural protein(n-MUC5AC),which treated with guanidine hydrochloride(GdnHCl),were added respectively to the above four macromolecular crowding reagent and dilute solution for renaturation.Then continuously monitored the light absorption and aggregation of both proteins at 488 nm by UV spectrophotometer at 25 degrees.The soluble protein concentration in supernatant was determined after centrifugation of the refolding end products,and the refolding yield of proteins in different refolding systems was analyzed.Results: The macromolecular crowding reagent with high mass concentration(? = 200 g/L)can reduce the refolding rate of protein by increasing the false aggregation of partial folding intermediates,while the macromolecular crowding reagent with relatively low mass concentration(? = 100 g/L)can result in good refolding of proteins.Meanwhile,in the same crowded reagent with the same concentration,the refolding yield of d-MUC5 AC was higher than that of n-MUC5 AC,while the degree of abnormal aggregation was lower than that of n-MUC5 AC.Conclusion: The macromolecular crowded environment intracellular can reduce the refolding rate of MUC5 AC in different degrees,and has a more significant effect on accelerate the misfolding and aggregation of glycosylated MUC5 AC.