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Protein Separation with Water-soluble Affinity Filtration Vector

Author: ChenPing
Tutor: HuSongQing
School: South China University of Technology
Course: Sugar works
Keywords: Cu2+-IDA-Dextran T2000 Lysozyme Recombinant AxCeSD Membrane separation
CLC: O629.73
Type: Master's thesis
Year: 2011
Downloads: 84
Quote: 1
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Abstract


Affinity ultrafiltration is a combination of affinity interaction which has the advantages of high specificity of affinity separation and membrane separation, which is with the advantages of easy to enlarge, no phase change and low energy consumption. The adsorption characteristics of Cu2+-IDA-Dextran T2000 with lysozyme and recombinant AxCeSD with 6 His-tags in the N-terminus were studied respectively. And the Cu2+-IDA-Dextran T2000 was adopted to separation of the recombinant AxCeSD from E. coli B384 cell disruption supernatant.The main contents are summarized as follows:Cu2+, Zn2+ and Ni2+ were used for the preparation of affinity filtration escort. The concentration of metal ions on the vector was detectived by atomic absorption spectrophotometry, Cu2+ was found to have the best adsorption and there is 0.8-1 Cu2+.on one monomer in the vector. Gel permeation chromatography （GPC） analysis showed that the molecular weight of vector was in the range of 236.47-2380.53kDa. It could be enough for separation of protein with MWCO 100kDa ultrafiltration membrane.The adsorption characteristics of Cu2+-IDA-Dextran T2000 with the lysozyme were studied. The results showed that pH and ionic strength had the significant effects on the adsorption capacity, the proper conditions for the adsorption of Cu2+-IDA-Dextran T2000 with lysozyme was: pH8.2, 0.2mol/L NaCl and reacted for 60min at 25℃. The adsorption of Cu2+-IDA-Dextran T2000 with lysozyme belonged to monolayer adsorption. To fit the Langmuir isotherm equation with experiment data, it was found that the theoretical maximum adsorption capacity of lysozyme was 13.65mg/g, dissociation constant of 2.068×10-4 mol/L.The adsorption characteristics of Cu2+-IDA-Dextran T2000 with recombinant AxCeSD were studied. The effect of pH, ionic strength, adsorption time and reaction temperature on the adsorption capacity were investigated. The results showed that pH8.0, 0.9mol/L NaCl and reaction time of 30min at 30℃were fit for adsorption. It was found that the adsorption of Cu2+-IDA-Dextran T2000 with recombinant AxCeSD also complied with monolayer adsorption. Similarily, to fit with the Langmuir isotherm equation, the maximum Cu2+-IDA-Dextran T2000 with theoretical adsorption capacity of recombinant AxCeSD was 125.15mg/g, dissociation constant was 3.259×10-6 mol/L. It is indicated that the adsorption of Cu2+-IDA-Dextran T2000 with recombinant AxCeSD has the characteristic of specific adsorption. Moreover, Cu2+-IDA-Dextran T2000 can be reused for adsorption of proteins.Phosphate buffer with the imidazole concentration of 0.25mol/L and pH8.0 was used as eluent for desorption of recombinant AxCeSD. The affinity filtration process was established for the separation of recombinant AxCeSD from E.coli B384 cell disruption supernatant with Cu2+-IDA-Dextran T2000, the recombinant AxCeSD was purified successfully under the detection of SDS-PAGE.

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CLC: > Mathematical sciences and chemical > Chemistry > Organic Chemistry > Natural compounds > α-amino acids,peptides, proteins, nucleic acids > Protein
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