Protein Modification And Separation By Using Environmentally-responsive Polymer

Separation and purification of proteins is an attractive and challenging task.Although the existing protein separation technology has met the need for purification and separation of most proteins,it still needs to face expensive cost and complicated operations.In addition,the separation of membrane proteins is still a bottleneck in proteomics.Therefore,the development of new,inexpensive,and highly efficient protein separation technologies is a long-term goal for people to explore.In this study,the thermo-sensitive Poly N-isopropyl acrylamide(PNIPAm)was polymerized in situ and reversibly grafted onto proteins by surface-initiated Atom Transfer Radical Polymerization(ATRP)technology.Developed a chemical selective thermal precipitation technique for protein separation and purification.Bovine serum albumin(BSA)was used as a thiol protein sample,and it was verified by SDS-PAGE that the chemical selective precipitation strategy can be applied to the separation and purification of BSA in a complex mixed system(including BSA-casein mixed system,Fetal bovine serum system,BSA-E.coli lysate mixed system).Using lysozyme as the non-sulfhydryl protein model protein,SDS-PAGE and LC/MS techniques proved that after the artificial introduction of sulfhydryl groups,chemical selective thermal precipitation technology can also be used for the separation of common proteins.Further study the feasibility of chemical selective thermal precipitation strategy in the separation and enrichment of prokaryotic membrane proteins.The ATRP technique was used to graft PNIPAm onto the surface of E.coli protoplast membrane proteins.Then the E.coli protoplasts were observed by field emission scanning electron microscopy.The results showed that there was no significant difference in cell morphology before and after a series of chemical modifications.Further characterization of the reversible fragmentation of the polymer confirmed that the initiated polymerization of PNIPAm occurred on the plasma membrane surface.Using chemical selective precipitation strategy,the membrane protein-enriched components were finally obtained and characterized by two-dimensional electrophoresis.In contrast to the reference group,12 relatively distinct protein spots(6 new protein spots and 6 protein spots with different concentrations)were selected for gel dot identification and mass spectrometry.The identification results were 8 membrane-associated proteins and 4 other proteins.The proportion of isolated membrane proteins was as high as 66.7%,which fully proved that the chemical selective thermal precipitation strategy can be effectively applied to the extraction and isolation of cell membrane proteins.This project developed a chemical selective thermal precipitation strategy based on reversible grafting polymers for the separation and purification of proteins.This method has a low cost and a large amount of purification and was expected to provide a new complement and choice to existing protein separation and purification technologies.