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Isolation and Purification Strategy of Target Protein in Binary-and Ternary-protein Mixture on Column Chromatography

Author: LiangXin
Tutor: BianLiuJiao
School: Northwestern University
Course: Biochemical Engineering
Keywords: Mixed protein samples Separation and purification Separation and purification strategies Ion exchange chromatography
CLC: Q51
Type: Master's thesis
Year: 2009
Downloads: 436
Quote: 4
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Abstract


This thesis abstracted as a two-component and three-component mixture of protein sample separation and purification of the target protein in the mixed samples of complex protein separation and purification process, ion exchange chromatography separation of the target protein in the two-component and three-component mixture of proteins The purification process objects to study the target protein in a chromatography column separation and purification strategy, and purification of high purity pig hemoglobin isolated from whole blood of the pig and remove the separation of low molecular weight hemoglobin purified from glutaraldehyde-polymerized porcine hemoglobin strategy, for example, to verify the separation and purification strategies proposed in this paper, the target protein to provide an efficient and viable solution for the pilot and large-scale separation and purification. Strategies in the separation and purification of the two-component mixing samples of pig hemoglobin and lysozyme CM SepharoseFast-Flow cation exchange chromatography separation and purification process, for example, their separation and purification strategy. Patterns, I mainly based on the effective capacity of the column chromatography medium using a flow-through mode elution, a small amount of protein concentrate enriched purpose. Tupu Type II main adsorption mode step gradient elution manner: Strategies in the separation and purification of the three-component mixing samples with pig hemoglobin, lysozyme and trypsin SP SepharoseFast-Flow strong cation exchange chromatography a separation and purification process for example, studied their separation and purification strategy. Three-component mixture separation and purification process can be summarized in three separation and purification strategy: purification strategy Ⅰ: purification step flow-through mode; purification strategy II: step gradient elution purification; purification strategy III: A two-step flow The wear mode purified. Obtained using the above separation and purification strategy pig hemoglobin and lysozyme by gel exclusion high performance liquid chromatography (SEC-HPLC) detection purity of more than 99.9%, the ion exchange high performance liquid chromatography (IEX-HPLC), detecting purity reached 95 % or more, and the recovery is more than 93%, and the target product can be different degrees of enrichment. Economic analysis, a new purification strategy than conventional purification strategies yield more than 10 times higher shortened single purified and expanded the yield per unit time. Types of buffer systems, optimization of the buffer system pH, salt ionic strength, dynamic column capacity conditions, as well as comparative adsorption mode and flow-through mode separation in the separation and purification of the purification of high purity pig hemoglobin in whole blood from the pig Strategies the purification results of the analysis, the final choice DEAE Sepharose Fast-Flow anion exchange chromatography medium for the stationary phase, and 20.0 mmol / L phosphate buffer 0.16 mol / L NaCl (pH 8.0) on the sample flow-through elution at impurity saturated chromatography column, and then to 20.0 mmol / L phosphate buffer solution of 1.0 mol / L NaCl (pH 8.0) eluting impurities. Purified Hb TSK-GEL G3000SWXL detect the purity of 99.99%, Shim-Pack WAX-1 detects a purity of 97.86% to electrophoretic homogeneity by SDS-PAGE and detected not detect phosphatidylserine and phosphatidylethanolamine, all targets were achieved the U.S. FDA the raw quality standards hemoglobin-based red blood cell substitutes. Porcine hemoglobin from glutaraldehyde-polymerized to remove the separation and purification of low molecular weight hemoglobin Strategies of them separately, in the Sephadex G-75 gel exclusion chromatography, exclusion limit for a hollow fiber column 10 kD ultrafiltration and DEAESepharose Fast-Flow anion exchange chromatography the three purification method on process route compared with the final choice of DEAE Sepharose Fast-Flow anion exchange chromatography. Optimized purified by chromatographic separation conditions: 20.0mmol / L PB 0.06 mol / L NaCl (pH7.3) as the equilibration buffer, a linear gradient elution with NaCl solution, and finally to 20.0 mmol / L PB 1.0 tool / L NaCl (pH7.3) and eluted. Aggregates purified hemoglobin by gel exclusion chromatography and multi-angle laser light scattering combined with detection of 64 kD molecular weight hemoglobin content of less than 5%, in line with the laboratory quality control standards.

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CLC: > Biological Sciences > Biochemistry > Protein
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