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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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【期刊论文】Refolding of denatured lysozyme assisted by artificial chaperones in reverse micelles
董晓燕, Xiao-Yan Dong, Xiao-Yue Wu, Yan Sun*
Biochemical Engineering Journal 31 (2006) 92-95,-0001,():
-1年11月30日
This article reports oxidative protein refolding assisted by artificial chaperones in reverse micelles formed by nonionic surfactant of sorbitan trioleate modified with Cibacron Blue F-3GA in n-hexane. The denatured/reduced lysozymewas used as a model protein and cetyltrimethylammonium bromide (CTAB) and-cyclodextrin as the artificial chaperones. The use of the artificial chaperones has proved to increase the refolding efficiency of denatured-reduced lysozyme at the concentration range studied (3.5-5.9 mg/mL). Moreover, the artificial chaperones increased the refolding yield in a wide range of urea concentrations. However, the optimal urea concentration range was little affected by the presence of the artificial chaperones.
Protein refolding, Lysozyme, Reverse micelles, Artificial chaperone, Nonionic surfactant, Cibacron Blue F-3GA
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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*
Biochemical Engineering Journal 28 (2006) 281-288,-0001,():
-1年11月30日
Sorbitan trioleate was modified with Cibacron Blue F-3GA (CB) to create an affinity surfactant and to form affinity-based reverse micelles in nhexane. The partitioning equilibria and the extraction kinetics of lysozyme and bovine serum albumin (BSA) were then examined. The solubilization capacity of the reverse micellar system for lysozyme increased linearly with increasing the CB concentration from 0.1 to 0.5 mmol L−1. In contrast, the capacity for BSA at 0.5 mmol L-1 of coupled CB was only about one-fifth that for lysozyme. It indicates a strong steric hindrance effect of the micelles for the high molecular mass protein. The overall volumetric mass transfer coefficient of lysozyme in the forward extraction increased from 0.43×10−3 to 1.25×10−3 s−1 with increasing CB concentration from 0.1 to 0.5 mmol L−1. Due to the high molecular mass of BSA, its volumetric mass transfer coefficient in the forward extraction was only one-sixth that of lysozyme. The ratio of the coefficient in the back extraction to that in the forward extraction was less than 0.03, much lower than those in other micellar systems. It indicates that the interfacial resistance in this system was severer than in others.
Affinity, Reverse micelles, Nonionic surfactant, Protein, Equilibrium, Extraction kinetics
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董晓燕, 王玮, 白姝, 孙彦
化工学报,2006,57(1):74-78,-0001,():
-1年11月30日
利用多角度激光光散射仪和圆二色谱仪研究小分子水溶性一元醇和多元醇对溶菌酶分子的稳定性以及对变性溶菌酶的空间构象和折叠复性的影响。发现在一元醇和二元醇溶液中溶菌酶分子的稳定性均随着醇浓度的增大而提高,而变性酶分子在醇溶液中形成过多的α螺旋和β折叠等二级结构不利于酶的折叠复性;只有较低浓度的多元醇和甲醇有辅助变性溶菌酶复性的作用,其中10%甲醇、20%甘油和30%乙二醇有利于促进酶的复性。
醇, 溶菌酶, 复性, 分子相互作用, 空间结构
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