Preperation Of A Modified Chitosan Affinity Membrane And Its Application In Purification Of His-Tag Protein

Affinity membrane chromatography possesses a great number of advantages,such as higher separation efficiency,more simple steps,larger-scale producation etc.It has promising prospects in the process of purification His-tag protetion.Developing suitable membrane material is great significance to affinity membrane application.In this paper,we developed a novel chitosan and polyvinyl alcohol chelated metal ion affinity membrane and studied the purification ability for His-tag protein.Specific contents are as follows.First,the membranes were crosslinked with epichlorohydrin and covalently coupled with iminodiacetic acid and silica gel was used to make pores.The micropore,functional groups,crystal structure of the modified membrane was analysised through SEM,FTIR,XRD,respecively.As the result showed,the pore size was 1.39 ?m when silica gel was 2 g.The functional groups?N-H2?were weaken and ?_N-H,?C=O were strengthen by epichlorohydrin activiting.The crystal structure had changed after modified.Elemental analyzer analysis showed that IDA connect amount was 3.79 mg/ g.Then Cu²⁺,Ni²⁺ was chelated on the modified membranes.The chelation capacity of Cu²⁺,Ni²⁺ in different pH,temperature,and initial metal ion concentration were studied.The results showed that the metal ion chelation amount was increased with the rising of temperature and metal ion concentration.the chelated Cu²⁺,Ni²⁺ optimal conditions were 35 ?,metal concentration 0.1 mol/L,NaCl 0.8,1.0 mol/L,pH 7.0,5.8.The maximum chelation ability were 98 mg/g,80 mg/g.Studies showed that the chelation of metal on modified membranes could be described by the Langmuir,Freundlich isotherm.The thermodynamic constants of phenomena: ?G0,?H0,and?S0were-8.57 kJ/mol?288 K?,-8.45 kJ/mol?288 K?;6.40 kJ/mol,15.91 kJ/mol;0.054 kJ?mol/K?,0.085 kJ?mol/K?respectively.The result shows that the chelation of metal on the modified membranes were spontaneous and endothermic.In order to study the purification capacity of the metal chelated ion affinity membrane,we choosed BSA and SHMT as target protein and optimized the purification conditions.BSA adsorption experiments showed that affinity membrane chelated Ni²⁺ had the maximum adsorption capacity of 14 mg/g.The optimum conditions were BSA initial concentration 1.2 mg/mL,NaCl 0.6 mol/L,pH 7.SHMT adsorption experiments showed that the 10.02 fold purification was obtained using chelated Cu²⁺ affinity membrane,the optimal loading conditions were 15 ?,0.6mol/L NaCl pH 8.The 9.01 fold purification was obtained using chelated Ni²⁺ affinity membrane,the optimal loading conditions was 4 ?,0.6 mol/L NaCl,pH 7.SHMT breakthrough curves showed affinity membrane had a good pore structure,SHMT elution peaks were obtained in elution process,SDS-PAGE results showed elution protein was a single band.In this work,we developed a novel macroporous chitosan polyvinyl alcoholt membrane.Iminodiacetic acid was successfully coupled on modified membrane,the chelated metal ion affinity membrane can reduce steric hindrance and increase adsorption capacity to protein.Then we optimized the conditions of chelation metal and purification his-tag protein.It provides a theoretical basis for large-scale purification his-tag proteins.