High Density Metal Affinity Functionalized Magnetic Graphene For Selective Separation And Purification Of Histidine-rich Proteins

The development of highly efficient and highly selective methods with high capacity for protein separation and purification is always one of the important aspects of proteomics research.In this paper,to resolve the drawbacks of poor selectivity and low adsorption capacity of graphene-based materials during protein separation and enrichment process,two functional magnetic graphene composites with high density metal affinity ligands were designed and synthesized.Two new approaches based on these two functional composites was presented for highly selective separation and purification of histidine-rich proteins with high capacity from complex biological samples.In chapter 1,a summery reviews the protein separation methods commonly used,outlines the principle and applications of metal affinity chromatography in protein separation and purification,as well as briefly describes the properties,preparation methods,surface functionalization of graphene-based materials and their recent progress in sample preparation field.In chapter 2,polylysine(PLL)was employed as a high-density ligand modifier to be functionalized on the surface of magnetic graphene(MG)through covalent bonding process.The composite was further conjugated with aspartic acid(Asp)by using glutaraldehyde as cross-linker,and then chelated copper ions to prepare a high density metal affinity-polylysine functionalized magnetic graphene composite(MGPLA-Cu).The obtained composite was characterized by FT-IR,XRD,TGA,VSM and SEM/TEM,and the results showed that the grafting density of metal affinity ligands in MGPLA-Cu composite was 14.1?mol m^-2.Owing to the strong metal affinity forces between the metal affinity groups and the residual histidine in proteins,the MGPLA-Cu composite exhibited high selective adsorption performance for histidine-rich proteins(hemoglobin).,A high adsorption efficiency of ca.99%was achieved for 200 mg L^-1 hemoglobin(Hb)in a phosphate buffer at p H 8,along with a maximum adsorption capacity of 334 mg g^-1.At the same time,the adsorption selectivity of Hb on the metal affinity magnetic graphene composite(MGA-Cu)without PLL modification was poor with a lower adsorption capacity of 227 mg g^-1.The retained Hb could be effectively recovered with a 0.2 mol L^-1 carbonate buffer containing 0.05 mol L^-1imidazole at p H 10,giving rise to a recovery of ca.97%.MGPLA-Cu composite showed favorable reusability,and high adsorption efficiency(>90%)could be maintained after five adsorption-desorption operation cycles.MGPLA-Cu composite was applied for isolation of Hb from human whole blood,SDS-polyacrylamide gel electrophoresis(SDS-PAGE)assay results confirmed that this method can be used for high selective isolation of Hb from complex matrix samples with high purity.In chapter 3,carboxymethyl cellulose was selected as the high-density modifier,and a metal affinity-carboxymethyl cellulose functionalized magnetic graphene,namely MGCI-Cu composite,was prepared by successive modifications of magnetic graphene with carboxymethyl cellulose(CMC),iminodiacetic acid(IDA)and then chelated with copper ions.The successful modifications of the graphene surface were demonstrated by various characterizations,and a high density of 6.17?mol m^-2 for metal affinity groups was obtained.The composite exhibited high adsorption selectivity toward histidine-rich proteins.The adsorption was governed by strong metal affinity binding force between hisitidine residues of proteins and immobilized Cu²⁺ions of MGCI-Cu composite.In particular,highly selective isolation of hemoglobin(Hb)was achieved in 0.2 mol L^-1 phosphate buffer at p H 8.The adsorption capacity of Hb significantly increased to 769 mg g^-1 in comparison to that of 435mg g^-1 on metal affinity modified magnetic graphene composite(MGI-Cu)without CMC modification.The adsorbed Hb molecules were recovered with a carbonate buffer(0.2 mol L^-1 p H 10)containing 0.5 mol L^-1 imidazole.MGCI-Cu composite displayed favorable reusability for at least four times after regeneration of the composite by edetic acid(EDTA)and Cu²⁺solution.The practical applications demonstrated that MGCI-Cu composite could highly selectively isolate Hb from human whole blood and polyhistidine-tagged recombinant protein from Escherichia coli(E.coli)lysate.