Preparation And Application Of Histidine-TAG Protein Separation Medium Based Chitosan

Protein is not only the basis of life activities, but also the functional macromolecule in living organisms. With the rapid development of biotechnolgy, numerous functional proteins expressed in microorganism use gene recombination technology, thereby making protein separation and purification prerequisite and ongoing.Chitosan, a natural non-toxic N-acetyl amino glucose composition of long chain polysaccharide, is the most abundant polymer after cellulose. Nowadays, it has been widely used in hydrophobic interaction chromatography (HIC) in recent years due to its good biocompatibility and chemical stability. Herein, EDTA which is a strong metal chelating ligand was introduced to the backbone of chitosan to give EDTA-chitosan. Subsequently, this polymer immobilized with metals (Ni2+, Cu2+, Zn2+) was used to the separation and purification of histidine-tagged proteins. Also, the resue and regeneration of this kind of polymer adsorbent was estibilished. The main experimental results are as follows:(1) Chitosan was used as the starting material, synthesised EDTA-chitosan, characterized and analyzed by fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), transmission electron microscope (TEM), thermo gravimetric analysis (TGA). Then the EDTA-chitosan was chelated with transition metal ions Ni2+, Cu2+ and Zn2+, respectively. The different adsorption time, initial concentration of metal ion and solution pH were investigated. The quantity of adsorbed metal ion was determined by inductively coupled plasma mass spectrometry (ICP-MS),The result showed that:EDTA groups were well grafted on chitosan, and when the adsorption was 3 hours,pH value was 4.0, the initial concentration of metal ions were 1.6 mg/mL, EDTA-chitosan could chelate the mostly metal ions.(2) The affinity properties with M2+-EDTA-chitosan and M2+-chitosan (M=Ni2+, Cu2+, Zn2+) were investigated using EGFP-(His)6-NH2 as the target proteins. Factors including incubation time, ionic strength, buffer pH, as well as the concentration of elution buffer were studied. The results showed that the adsorption equilibrium was reached in 40 min, pH and ionic strength could change the combination of electrostatic and hydrophobic interaction, which affected the purity and content of target protein.(3) Studied the isolation and purification of EGFP-(His)6-NH2 and Kana-(His)6-NH2 expressed by E.coli. The results show that when the pH of buffer was 7.2, the ionic strength was 500 mmol/L NaCl, and the elution buffer solution containing 500 mmol/L imidazole, which obtained the target protein EGFP-(His)6-NH2 of 95% purity; and the pH of buffer was 6.2, the ionic strength was 500 mmol/L NaCl, and the elution buffer solution containing 200 mmol/L imidazole, which obtained the target protein Kana-(His)6-NH2 of 90% purity.(4) The reuse ability and regeneration methods of the purifing medium were studied. The results showed that:M2+-EDTA-chitosan (Cu, M=Ni, Zn) showed a prominent reuse performance, in the first 5 times of reuse, the binding rate of the protein only showed a trace of the decline, respectively,3.9%(Ni2+-EDTA-chitosan).3.3%(Cu2+-EDTA-chitosan)、 2.8%(Zn2+-EDTA-chitosan) compared with the first use, the use of 2 mol/L HNO3 desorption and reloading of metal ions on the ligand, continue to be used for the affinity of the protein, its affinity ability could be a good recovery.(5) EDTA-chitosan was used to form membrane for the purification of EGFP-(His)6-NH2 and Kana-(His)6-NH2. The results showed that membrane purification exhibited great advantages compared with directly used for the purification of his-tagged proteins, which shortened the separation time and saved cost in a great extent.