Affinity Precipitation Systems With PH/Thermo-Responsive Polymer In Protein Purification And Its Molecular Interaction Mechanism Research

The development of bio-separation methods is to get protein from the broth simply and efficiently.The simplicity needs less procedures and the efficiency means this method should specificly purify target protein from the impurities.Affinity precipitation is the combination of precipitation and affinity purification,with the advantages of simple,recoverable and selective to the target protein.Two affinity precipitation systems were built to purify transglutaminase and porcine circovirus type ? Cap protein,respectively.The first process was the preparation of affinity polymers.The pH-responsive polymer(PMMDN)was consisted of methacrylic acid,dimethylaminoethyl methacrylate,methyl methacrylate and N-methylol acrylamide.The monomer molar ratio was 9.5:3.8:7.2:1.0.The isoelectric point was 4.76.Affinity polymer(PMMDN-T)was obtained by L-thyroxine attached to PMMDN.When the ligand density was 51.22 mol/g,the isoelectric point was 4.21 and the recoveries of PMMDN-T were above 95.0%.The thermo-responsive polymer(PNDBD)was also composed of N-isopropylacrylamide,dimethylaminoethyl methacrylate,butyl methacrylate and N-methylol acrylamide,with monomer molar ratio(14.2:2.4:0.6:1.0),the lowest critical solution temperature of 34.2?.The connection of nickel ions to PNDBN was achieved by iminodiacetic acid.When the ligand density of PNDBN-Ni was 49.23?mol/g,the the lowest critical solution temperature was 36.1?and the recovery was about 94.0%.The properties of the polymer were detected by a variety of characterization methods.The surface characteristics of PMMDN-T and PNDBN-Ni under scanning electron microscope were rougher than that of PMMDN and PNDBN in the direct sample preparation.The pores observed on PMMDN-T and PNDBN-Ni were smaller than PMMDN and PNDBN-Ni within the precipitation dispersion method.The affinity polymer with two kinds of sample preparation were also observed under the transmission electron microscope.It was found that PMDBN and PNDBN-Ni were granulated in the preparation of precipitated dispersion method.The infrared spectroscopy could be used to analyse the characteristic functional groups of the monomers in the affinity polymer.The photoelectron spectroscopy was used to detect the composition of the elements in the polymer and verify the presence of the characteristic elements in the affinity ligand.The gel permeation chromatography could measure the molecular weight of the PMMDN,PMMDN-T,PNDBN and PNDBN-Ni and the value were 4.1x106,7.6x105,3.1x 105 and 2.1x106,respectively.Dynamic light scattering was used to measure the particle size of the polymer.The diameter of PMMDN-T(198.3 nm)was smaller than it of PMMDN(282.3 nm).and PNDBN-Ni(90.1 nm)has a larger particle size than it of PNDBN(33.3 nm).Through the specific results from these characterization methods,the polymerization and ligand connection processed could be conducted in a more targeted way.The effects of pH,temperature,ionic strength and ligand density on the adsorption process were studied by response surface methodology during the purification of tranglutaminase with pH-responsive affinity polymer(PMMDN-T).The optical adsorption conditions were pH 7.00,NaCl=0 mol/L,ligand density with 50 ?mol/g and 30?.The optical elution condition was pH 10.0 Gly-NaOH.The elution ratio and enzyme activity retention ratio were 98.44%and 92.19%,respectively.The adsorption isotherm at different temperatures were made and the Langmuir-Freundlich model was applied.The theoretical adsorption capacity at 30.0? was 4.67 ?mol/g.The purity of the sample detected by the electrophoretic detection was similar to that of the commercial transglutaminase.In the purification of porcine circovirus type ? Cap protein with PNDBN-Ni,the pH,ionic strength and affinity density of the adsorption were optimized.At pH 6.0,NaCl with 0 mol/L and ligand density with 56.22 ?mol/g,the adsorption capacity was 36.81 mg/g.The elution process was carried out with 0.01 mol/L Na2SO4 and 0.1 mol/L guanidine hydrochloride.The elution amount was 1.02 mg/mL.The result of adsorption time assay showed that the adsorption process is closed to the quasi-first-order kinetic model and the Langmuir model is selected for the adsorption isotherm.The final purification results were detected by protein electrophoresis and the results of the elution had less bands than the samples of fermentation broth.Finally,the interaction of the affinity ligand and protein was studied by molecular docking technique and instrumental analysis.The DOCK6 software was used to dock L-thyroxine and transglutaminase.After that,nine edible pigment molecules were respectively docked with transglutaminase.And the result of crocein orange G was superior to other pigment molecules.The docking of L-thyroxine and glutamine transaminase was simulated by AutoDock software,which verified with the results of DOCK6,and the results were graphical.The interaction between the affinity ligand and protein was also studied by turbidity method,Zeta potential method,low field nuclear magnetic resonance method and isothermal titration calculation method.The X-ray photoelectron spectroscopy could detect the state change of nickel in the polymer to study the interaction of metal ions and protein.The combination of molecular docking and instrumental analysis could illustrate the interatction mechanism and process about the protein and L-thyroxine.Moreover.it also could be a new method for ligand screening.In this paper,the effects of two different affinity precipitation systems on protein purification were studied.And the process of affinity was studied by molecular simulation and instrumental analysis.The aim of this study was to improve the general process of purification of protein by affinity precipitation,which could benefit its application in more protein purification.