Preparation Of Functional Magnetic Nanoparticles And Their Application In Separation And Purification Of Histidine-tagged Proteins

In the field of protein separation and purification,histidine-tags are widely used.There are currently many methods for separating and purifying recombinant proteins carrying histidine-tags,but these methods usually have some limitations,such as complicated preparation of adsorption materials,low adsorption capacity,high cost,or complicated procedures for separation and purification.Therefore,it is of great significance to develop a simple and efficient method for histidine-tagged protein separation and purification.Using a facile preparation method,in this study,we obtained two functional magnetic nanoparticles,which were nano magnetite coated by sodium citrate chelating Ni²⁺(NM-SC-Ni)and nano magnetite coated by sodium bitartrate chelating Ni²⁺(NM-SB-Ni).We demonstrated that the two nanoparticles could successfully achieve specific adsorption and efficient purification of histidine-tagged proteins in complex cell lysates.The study includes the following three parts:(1)Preparation of NM-SC-Ni and NM-SB-Ni magnetic nanoparticlesFe₃O₄ magnetic nanoparticles(Fe₃O₄-MNPs)were successfully prepared by co-precipitation method.X-ray powder diffraction method was used to confirm the synthesized magnetic nanoparticles.Transmission electron microscopy revealed that the size of Fe₃O₄-MNPs was about 10-20 nm.Sodium citrate and sodium bitartrate were coated on the surface of Fe₃O₄-MNPs by ultrasonic method,then NM-SC and NM-SB were obtained.Based on the coordination binding of carboxyl group and Ni²⁺,NM-SC-Ni and NM-SB-Ni magnetic nanoparticles were further prepared,respectively.The results of thermogravimetric analysis and X-ray photoelectron spectroscopy analysis demonstrated that the two materials were successfully obtained.In addition,NM-SC-Ni and NM-SB-Ni exhibited good dispersibility and magnetic separation effect.(2)Preparation of histidine-tagged proteinsWe selected two different recombinant proteins(his-h CA II and his-Pin1)carrying histidine-tags as research models for the subsequent detection of NM-SC-Ni and NM-SB-Ni nanoparticles in the application of separation and purification for histidine-tagged proteins.The construction of E.coli recombinant plasmid expressing his-h CA II protein and the optimization of expression conditions had been completed in our laboratory.In this part of the work,we constructed the his-Pin1 E.coli expression plasmid and determined its optimal expression conditions:the concentration of Isopropyl-?-D-thiogalactopyranoside(IPTG)inducer was 0.2 mmol L^-1,the induction temperature was 37?,and the induction time was 6 hours.In conclusion,we successfully obtained E.coli cell lysates expressing his-h CA II or his-Pin1 recombinant proteins.(3)Application of NM-SC-Ni and NM-SB-Ni in separation and purification of histidine-tagged proteinsUsing E.coli cell lysate containing his-h CA II or his-Pin1 recombinant proteins as the research objects,we systematically evaluated the ability of NM-SC-Ni and NM-SB-Ni for specific adsorption and separation of histidine-tagged proteins,and also tested the adsorption capacity and reusability of these two magnetic nanoparticles.The results showed that,in the complex cell lysate,NM-SC-Ni and NM-SB-Ni could achieve the selective adsorption and separation of histidine-tagged proteins,and the targeted proteins obtained had higher purity.The isotherm adsorption curve was fitted to Langmuir model,and showed that the saturated adsorption capacities of NM-SC-Ni and NM-SB-Ni to purify his-h CA II protein were 625 mg g^-1 and 556 mg g^-1,respectively.The results of polyacrylamide gel electrophoresis showed that the separation and purification ability for the histidine-tagged proteins did not change significantly when the two materials were reused for 6 times,indicating the excellent stability and recyclability of these two nanoparticles as an adsorbent.In summary,this study successfully obtained two nickel chelated magnetic Fe₃O₄nanoparticles coated with sodium citrate or sodium bitartrate using a simple preparation method.The results showed that the raw materials for preparation of the above two nanoparticles were cheap and easy to obtain,and the two nanoparticles could specifically and efficiently separate and purify of histidine-tagged proteins from complex samples by magnetic separation method,which provides a simple,economical,and efficient method with promising application prospects for the extraction and purification of histidine-tagged proteins.