Preparation And Application Of Water-soluble Affinity-ultrafiltration Vector Of Recombinant Protein

Affinity-ultrafiltration is combined the high selectivity of biological affinity and membrane separation which is with the advantages of fast and easy to enlarge. It has a broad prospect for industrialization. In the paper, DextranT2000 is repared as the water-soluble affinity-ultrafiltration vector matrix. It was characterized by epoxy activation, coupled with iminodiacetic acid (IDA), chelated copper, prepared as water-soluble affinity-ultrafiltration vector and applied to the separation of the recombinant protein AxCesD. The main contents of this paper and the results are summarized as follows:1,The epoxy activation of dextran T2000. By studying the concentration of alkali treatment, alkali treatment time, epichlorohydrin concentration, ultrasonic intensity, reaction time, reaction temperature in the epoxy activation of dextran T2000 the best conditions are as follows: the concentration of 4% dextran T2000 in the concentration of 0.1mol/L of NaOH solution after reacting for 30min, the alkali activation of dextran T2000 is got and then with 4% (V/V) of epichlorohydrin, in ultrasound conditions at frequency 130kHz, power 500W, 30℃reaction for 4h, the concentration of its epoxy is 3.3472mmol/g.2,The synthesis of water-soluble affinity-ultrafiltration vector. The epoxy activated dextran T2000 was affinitied with iminodiacetic acid (IDA) and chelated copper in that methods and conditions: in ultrasound conditions at frequency 130kHz, power 500W, 30℃reaction for1h and the epoxy activated dextran T2000 coupled IDA was saturated and then chelated copper ions, the water-soluble affinity-ultrafiltration vector was got with its copper-chelating capacity of 2.5806mmol/g.3,Analysis the epoxy activated dextran T2000 by infrared spectroscopy and in the wave number 1270cm-1 and 870cm-1 it has a specific absorption peak of epoxy; by high performance gel chromatography, the molecular weight of the water-soluble affinity-ultrafiltration vector is about 88-500kD.4,Purify the recombinant protein AxCesD by column chromatography to study the separation characteristics of the water-soluble affinity-ultrafiltration vector. The average absorption rate of water-soluble affinity-ultrafiltration vector is 97.36% for the recombinant protein AxCesD and the average elution rate is 95.44%. Separate the recombinant protein AxCesD from the cells liquid by the water-soluble affinity-ultrafiltration vector and detect the recombinant protein AxCesD by SDS-PAGE, the high purity of the recombinant protein AxCesD is got.