Research On Affinity Precipitation Mechanism And Applications In Protein Purification

Protein purification on a large scale is still a challenge nowadays.Affinity precipitation was reported as a potential technique for the purification of proteins at early stage of downstream processing. The technique could be achieved by using reversibly soluble-insoluble polymers coupled with ligand as an affinity carrier to purify proteins from large volume of dilute solution material. The key of affinity precipitation is the syntheisi of the response polymers. In this study, pH-response and thermo-response polymers were synthesized. Methyl acrylate, methacrylate, dimethylaminoethyl methacrylate, butyl methacrylate and N-methylol acrylamide were used to synthesize the pH-response polymer PMMDN, IR was used to analyze its structure. The pI of PMMDN was4.51obtained by measuring the Zeta potential. The molecular weight was6.78×104, whick was tested by gel chromatography technique. The multiple recoveries were higher than96.0%. N-isopropyl acrylamide, butyl acrylate and N-methylol acrylamide were used as monomers to synthize the thermo-response polymer PNBN by random polymerization. The lowest critical temperature of the polymer was30.8℃measured by the spectrophotometer. Its molecular weight was3.17×104, and the recovery of PNBN was99.8%with adding0.5mol/LNaCl.There are four different ligands were connect to two polymers. PMMDN was connected Cibacron Blue F3GA(PMMDN-CB), metal ions(PMMDN-IDA-Cu2+) and L-thyroxine ligand (PMMDN-T); PNBN was connected to the L-thyroxine(PNBN-T).The pI of these different ligand to the pH-response polymer were4.45,4.35and4.65, respectively. The recoveries of these polymers were all higher than96.0%. The LCST of PNBN-T was32.8℃,five times recoveries of PNBN-T were99.0%in the presence of NaCl.Subsequently, the obtained four different affinity precipitation polymers were applied to purify cellulase, endoglucanase. lysozyme, human serum albumin and glutamine transaminase. Molecular simulation technology, infrared spectroscopy, circular dichroism, biological macromolecules interaction instrument (Octet), SDS-PAGE electrophoresis were all used in this study. Cellulase was purified by PMMDN-CB. Optimal adsorptions could be achieved at pH7.2and1.0mol/1NaCl. FTIR confirmed that ligand CB was immobilized on the polymer and cellulase was adsorbed on PMMDN-CB. The optimal eluant was0.1mol/1pH7.1Tris-HCl with20.0%glycol. The elution recovery of total cellulase activity and endo-glucanase activity was almost84.4%and99.8%, respectively. The SDS-PAGE showed the main bands with a molecular weight corresponding to that of the native cellulase. Molecular docking by DOCK software was used to calculate and help to determine the species of the metal chelating agent and a metal ion ligand. Iminodiacetic acid was chose as metal chelating agent for its stability and activity, copper ions was decided because it could be combined with protein stably.All experiments datas tesefy the simulation results. Use PMMDN-IDA-Cu2+to separation endoglucanase in the conditon of pH5.0,1.0mol/L NaCl concentration and30℃in two hours to reach equilibrium; the eluant was0.5mol/L imidazole with l.Omol/L guanidine hydrochloride, and the elution ratio of protein and endonuclease activity were up to98.5%and99.3%, respectively. Use PMMDN-T as affinity polymer to get lysozyme from salted duck eggs. The optimal adsorption condition was pH5.5with some NaCl to increase ionic strength, ligand density was60.0μmol/g. The maximum adsorption capacity of lysozyme and the dissociation constant of the polymer obtained by adsorption isotherm22.8mg/g and0.085mg/g, the Octet analysis also confirms the correctness of the results. Circular dichroism proved that L-thyroxine had affinity force to lysozyme. pH7.0PB with0.5mol/LUrea was used as the eluent to obtain higher efficiency, the elution ratio of protein and enzyme activity up to85.0%and95.4%, respectively. L-thyroxin which was first used as affinity ligand was immobilized on the PNBN for affinity precipitation of HSA. The optimal adsorption condition was0.02M Tris-HCl buffer (pH7.0) and the adsorption capacity of HSA on the polymer was14.9mg/g polymer in affinity precipitation process. Circular Dichroism spectra and ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The elution recovery of total HSA was93.6%by1.0mol/L NaSCN. When the affinity polymer was applied in the purification of HSA from human serum, the SDS-PAGE showed that a purified HSA single band was obtained.PNBN-T was used to extract pure glutamine transaminase (TG) from the fermentation broth, the optimum adsorption conditions was pH5.0, ligand density for59.5.0μmol/g, shaken at15°C for two hours to achieve maximum efficiency. The maximum adsorption capacity and dissociation constant were169.4mg/g and1.35mg/g, respectively. The Octet experimental verified the correctness of the results. Circular dichroism proved that L-thyroxine make the secondary structure of TG change by the affinity force between. The optimum elution condition was pH10.0Gly-NaOH, and the elution ratio of protein amount and activity were up to99.01%and95.85%, respectively. The SDS-PAGE was used to analyze the final results, and the electrophoretically pure TG was obtaind.