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【期刊论文】Modeling and Simulation of Fed-Batch Protein Refolding Process
董晓燕, Xiao-Yan Dong, Guang-Quan Shi, Wei Li, and Yan Sun*
Biotechnol. Prog., 2004, Vol. 20, No.4,-0001,():
-1年11月30日
The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7mg mL-1, respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.
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董晓燕, Haiqing Yu, Xiao-Yan Dong, Yan Sun*
Biochemical Engineering Journal 18(2004)169-175,-0001,():
-1年11月30日
An efficient procedure for the selection of high affinity clones from a heptapeptide phage display library was developed. Lysozyme was used as a model protein to demonstrate the selection strategy. Effect of bovine serum albumin (BSA) concentration on screening the phage library was discussed and proper BSA concentration on plate blocking was determined. The elution procedure was improved by alternatingly eluting the bound phages with glycine-HCl buffer (pH 2.2) and high-concentration target protein solution. The modified method was compared with others including the conventional protocol, and the results confirmed that the modified procedure could yield high affinity phages that might be lost by other screening methods. Through comparison of the DNA sequences of foreign peptides of the clones showing specificity to lysozyme molecules, the HWWW motif was found to be the necessary amino acid sequence for the affinity. The electrostatic and hydrophobic interactions are considered to contribute to the affinity for the protein. Moreover, protein chromatography with the immobilized HWWWPAS on Sepharose gel indicated the strong binding affinity of the peptide for lysozyme.
Phage display library, Peptide, Ligand, Selection, Lysozyme, Affinity chromatography
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董晓燕, Hai-Qing Yu, Xiao-Yan Dong, Yan Sun
Chromatographia 2004, 60, October (No.7/8),-0001,():
-1年11月30日
A heptapeptide phage display library was screened with insulin to find its ligands for affinity chromatography. The peptide was synthesized and coupled to EAH Sepharose 4B (5.4umol mL-1 bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6mg mL-1 bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin.
Column liquid chromatography Affinity chromatography Peptide ligand Phage display library Insulin
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董晓燕, Yang Liu, Xiao-Yan Dong, Yan Sun*
Journal of Colloid and Interface Science 290(2005)259-266,-0001,():
-1年11月30日
The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. The reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate were extensively characterized in water content, hydrodynamic radius, and aggregation number. The results show that the water content and hydrodynamic radius of the reversed micelles were significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate had a wider aggregation number distribution than the Span 85 reversed micelles. Using lysozyme as a model protein, protein solubilization by the reversed micelles was investigated. Lysozyme solubilization increased significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. High solubilization of lysozyme was obtained by the affinity-based reversed micelles of 62.7mmol/L Span 85 with coupled CB higher than 0.25mmol/L. Lysozyme recovery was carried out using a stripping solution of high ionic strength. The recovered lysozyme exhibited an activity equivalent to the native lysozyme and its secondary structure was also unchanged. The results indicate that the reversed micellar system would find potential application in protein separation.
Affinity-based reversed micelles, Span 85, Cibacron blue F-3GA, Water content, Hydrodynamic radius, Solubilization, Lysozyme
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【期刊论文】Kinetic model of lysozyme renaturation with the molecular chaperone GroEL
董晓燕, Xiao-Yan Dong, Yu-Bing Wang, Xiao-Guang Liu & Yan Sun, *
Biotechnology Letters 23: 1165-1169, 2001.,-0001,():
-1年11月30日
From the renaturation kinetics of denatured/reduced lysozyme assisted by the molecular chaperone GroEL, a simplified kinetic model was established based on the competition between protein folding and aggregation. In the presence of GroEL and ATP, the aggregate formation was a second order reaction. With 2mM ATP, a renaturation yield of 90% at a high renaturation rate was obtained when the molar ratio of GroEL to lysozyme was 1: 1.
GroEL,, kinetics,, lysozyme,, model,, renaturation
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