Synthesis Of Novel Metal Affinity Chromatography Materials For Highly Specific Separation Of Histidine-tagged Proteins

As a kind of important biomacromolecules,protein plays crucial role in various life activities.Easy separation and purification of proteins are fundamentally important to understand biological systems.Histidine tag?His-tagged?proteins play important roles in various physiological processes,such as cellular metal homeostasis,detoxification,antimicrobial response,and intrinsic pathway of coagulation.And his-tagged recombinant proteins are widely employed during the protein engineering.Besides,the low-abundant biomarkers are always submerged by the high-abundant His-tagged proteins in biomedical analysis.Thus,the separation of His-tagged proteins from biological samples plays a crucial role in the proteomic and biomedical analysis.Traditionally,immobilized metal affinity chromatography?IMAC?and metal oxide affinity chromatography?MOAC?have been considered as one of the most important and effective approaches for His-tagged proteins separation among current methodologies.However,the inherent disadvantages of conventional IMAC and MOAC materials are also obvious,such as affinity ligands used and limited surface area?low surface metal ion density?as well as laborious synthesis routes,which restricts their practical application in His-tagged protein separation.Therefore,the development of innovative IMAC?MOAC?materials with an excellent adsorption capacity,convenience for preparation as well as good recyclability remain of great interest.In this thesis,three kinds of novel IMAC?MOAC?materials with easy preparation and great adsorption capacity were fabricated for the selective capturing of his-tagged proteins.The main contents are as follows:1.Hollow Co₃O₄/NiCo₂O₄ nanoparticles?HCNs?were synthesized with zeolitic imidazolate framework-67?ZIF-67?nanocrystals as the templates.Uniform zeolitic imidazolate framework-67/Ni-Co layered double hydroxides yolk-shelled structures are first synthesized and then transformed into HCNs by thermal annealing in air.The prepared HCNs composites were fully characterized by powder X-ray diffraction,Scanning electron microscopy,fourier transform infrared and X-ray photoelectron spectroscopy analysis.HCNs possess high surface metal ion density and used as affinity adsorbent to directly enrich and separate His-tagged protein.HCNs exhibited large adsorption capacity for bovine hemoglogin?about 1000 mg g-1?and showed excellent recyclability?5 times?with high recovery?80%?.The good results demonstrate the potential of HCNs composites in the separation of histidine-rich proteins.2.The aim of this part was to develop a reliable method to fabrication of copper oxides for separation hemoglobin.To accomplish this goal,nano plumose copper oxides?NPCuO?were facilely synthesized via a simple and economical wet-treatment method using Cu-based metal organic framework?HKUST-1?as precursor.By taking advantages of the strong affinity interaction between copper???ions and histidine of hemoglobin,NPCuO possesses high absorption ability toward hemoglobin?more than 2000 mg g-1?.The results showed that NPCuO can be potentially used for removing abundant protein in proteomics.3.A copper ions functionalized magnetic nanostructured porous carbon material?Cu-Fe/C?was easily synthesized using a microwave enhanced high-temperature ionothermal method with starch,FeCl₃ and CuSO₄·5H₂O as precursors.The prepared Cu-Fe/C composites were fully characterized by powder X-ray diffraction,Scanning electron microscopy,fourier transform infrared and thermogravimetric analysis.Then,Cu-Fe/C were developed as a new nonligand modified strategy to selectively capture bovine hemoglogin?BHb,a classical His-tagged protein?,results display a selective protein adsorption capacity to efficiently capture BHb.