Affinity Partition of Acid Proteases in Aqueous Two-Phase Systems: Modeling and Protein Purification

[摘要] References(16)Aqueous two-phase systems consisting of dextran and hydroxypropyidextran (HPD) were used in protein partition studies. The phase diagram of such systems was determined by fluorescein-labelled dextran or HPD. The phase envelope was close to symmetrical, consistent with the Flory-Huggins theory for polymer solutions owing to the similarities between the two phase-forming polymers. A new mathematical model for affinity partition was developed. Simulation results from this model indicated that the ratio of dissociation constant of protein-ligand complex to total ligand concentration should be lower than 10–3, and the ratio of total protein concentration to total ligand concentration should be kept below 1 to get the best partition results. Partitions of pepsin, chymosin, and Endothia parasitica protease were studied in affinity dextran/HPD aqueous two-phase systems with pepstatin attached to dextran as the ligand. The proteins strongly preferred the bottom phase into which the pepstatyl-dextran segregated. Data from the partition experiments can be satisfactorily correlated with the model developed. Purification of chymosin was carried out in this affinity system with a five-step liquid-liquid extraction. The purification factor was 6.2 with a yield of 83%.

[关键词] Aqueous Two-Phase Systems;Affinity Partition;Acid Proteases;Protein Purification