Dissertation > Excellent graduate degree dissertation topics show

Study on In-vitro Refolding of Lysozyme and Recombinant Human Interferon-γ

Author: GaoYongGui
Tutor: YaoShanZuo
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Protein in vitro refolding Renaturation Lysozyme Disulfide Fed-batch operation Size exclusion chromatography refolding Refolding kinetics model Recombinant human IFN-γ Inclusion bodies Small molecular chaperone GroEL (191-345) (Mini-GroEL)
CLC: Q51
Type: PhD thesis
Year: 2002
Downloads: 443
Quote: 10
Read: Download Dissertation

Abstract


So far, little is known about the people convinced that the existence of a second genetic code - protein folding password. How high concentration of inclusion body proteins, high-yield complex active products often face a difficult problem, which has become one of the bioengineering industry bottleneck. To this end, this paper on the model protein lysozyme and genetically engineered drug human interferon-γ inclusion body protein refolding refolding study, in order to promote an early settlement of this problem. First lysozyme as the model protein, through the influence of the reducing agent dithiothreitol (DTT) of a free thiol group in its refolding process and renaturation, found that if the protein concentration does not exceed 40 mg / mL, the denaturing solution DTT optimal concentration 30mM, so direct refolding without removal DTT, simplifies subsequent refolding process. The degree of degeneration of lysozyme refolding effect, so the study of lysozyme suitable denaturing conditions, to achieve the purpose of improving the refolding yield. Refolding method is to study other methods. Direct refolding law by the high concentration of lysozyme, the optimum GSH concentration of 6 mM GSH: GSSG compared with 2 to 5, in addition to need to add 3 ~ 4M urea. Within this range, suitable urea concentration slightly increases with the increase in the protein concentration. Compared with step refolding method, the step flow plus the refolding of denatured lysozyme operation is not only able to obtain higher final concentration of the refolding, and the refolding yield increased by 10% to 15%, and reduced the urea concentration. Then focused on a size exclusion chromatography refolding of lysozyme conditions, including column, the amount and proportion of GSSG with GSG, urea concentration, flow rate, sample volume (on a like manner and on the kind of volume), and protein concentration. First to investigate the successive loading operation of the refolding method of size exclusion chromatography, and operation performed with a single sample, the activity of both yield no difference, but the improved effectiveness of about 25% is a continuous operation, but also saves refolding buffer. On the basis of the proposed kinetic model and the model of the denatured protein in vitro refolding process equation solving using C program model equations and experimental data for lysozyme refolding fit, fit coefficients R is basically between 0.98 to 0.99, indicating that the kinetic model can be well described in vitro refolding of lysozyme. Add urea refolding solution so that the rate constant K 2 and K 3 value decreases renaturation time, but increased the K 2 / K 3 , have a stronger role of urea on the aggregation reaction, this is the right amount of urea to promote the mechanism of lysozyme. Fitting the urea concentration and rate constant K 2 and K 3 mathematical expressions, and combined with the model equations, for the first time to explore how theory of folding complex resistance to predict the yield as well as to optimize the refolding conditions and select the optimal urea concentration is based on the protein concentration, thus provides an important theoretical guidance for renaturation methods and industrial design of the refolding process. Using a combination of single factor and orthogonal experiment method, washing and purification of recombinant human IFN-γ inclusion bodies, eventually IFN-γ inclusion body purity of 80% or more, about 15% of the target protein only loss. The summary of the doctoral thesis of Zhejiang University, and then examine the size exclusion chromatography refolding IFN-Y inclusion bodies, the results show that the technology not only in vitro refolding of denatured IFN-Y of inclusion bodies, and the target protein purified. Refolding IFN-Y of inclusion bodies of a size exclusion chromatography method on the basis of IFN-Y activity was measured in the laboratory to establish and obtain the specific activity of 6.09 gi 06 IU / mg, concentration of 0 .24 mg / mL IFN-v sample method was 82.1%. Finally, proposed of activity IFN1 from genetically engineered bacteria pBV220IFN1DH5a production process. New small sub-study on the basis of the partner Mini-GroEL, free small molecular chaperone assisting positive inclusion bodies of the N-Y refolding prepared in the laboratory. According to the small molecular chaperone Histidine tag features, for the first time experimental device designed Ni-NTA affinity and immobilized small sub-partner column refolding positive N-Y of inclusion bodies. The results showed that the device not only avoid denaturant combined damage to small molecular chaperones and immobilized and continuously repeated 4 times, refolding activity of IFN-Y size did not change significantly. Use the device renaturation small N-Y inclusion bodies, one injector 0.98mg denatured IFN-Y of inclusion bodies, available after the immobilized molecular chaperone column activity up to of 6.4X 10 spoon U / mg concentration of 0. sample of of 14mg/mL the IFN-Y, the operation is very convenient.

Related Dissertations

  1. Roles of MMP-7 and Lysozyme in the Pathogenesis of Ulcerative Colitis Induced by DSS in Balb/c Mice,S858.91
  2. Chemical Synthsis, Activity Evaluation and Expression of the Novel Conotoxins from South China Sea,TQ464
  3. NO Intervention of Carbon Disulfide Toxicity on the Reproductive Secretion Function of Hypothalamus -pituitary-testis Axis in Male Rats,R363
  4. Related Risk Factors to Caries after Nasopharyngeal Carcinoma Radiotherapy,R739.63
  5. Effect of Diallyl Disulfide on Cell Proliferation of Human Leukaemia K562 and Expression of Bcl-2, Bax, Survivin,R733.7
  6. The Mechanism of Cycle Arrest and Apoptosis in Human Glioblastoma U251 Cells Induced by Diallyl Disulfide,R739.4
  7. Study of the Effect and Mechanism of Protein Disulfide Isomerase in the Pathogenesis of Nonalcoholic Fatty Liver Disease in Vitro,R575.5
  8. Prokaryotic Expressionand Purification of MMP9-PEX and Research of Its Renaturation,R346
  9. Research on Augmentation of Sludge Internal Carbon Release and Removal of Nitrogen and Phosphorus in Hydrolysate,X703
  10. Study on Lysozyme Crystallization Induced by Ionic Liquids and Its Mechanism,O629.8
  11. Preparation of Temperature-responsive Monolith Column and the Study of Its Chromatographic Performance,O657.7
  12. The Study on Transformation of Dual Genes Lyz-GFP in Medicago Sativa,S541.9
  13. Branched Polyethylenimine Derivatives with Reductively Cleavable Periphery for Safe and Efficienct in Vitro Gene Transfer,R346
  14. Study on the Expression of Recombinant Human Lysozyme Fusion Gene in Pichia Pastoris,Q78
  15. Preparation and Tribological Properties of Tungsten Disulfide (WS2) Film,O313.5
  16. Fabrication of Antibacterial Wool Surfaces Via Layer-by-layer Electrostatic Self-assembly Immobilization of Lysozymes,TS195.58
  17. Effects on Physicochemical and Microbial Characteristics of Lupin-Milk Treated by Ultra High Pressure Homogenization and Combined Effects of Ultra High Pressure Homogenization and Lysozyme on Microorganism,TS214.2
  18. On-line Isolation of Lysozyme from Egg White Using Multi-walled Carbon Nanotubes,TS201.25
  19. Development of Desulfurization Technique for Improving Removal Efficiency of Mercaptans from Liquefied Petroleum Gas (LPG),TE624.5
  20. MD Simulation Research on Interations between Water Clusters and Protein,O629.73
  21. Study on Properties of FeS2 Prepared by Hydrothermal Method,TM912.9

CLC: > Biological Sciences > Biochemistry > Protein
© 2012 www.DissertationTopic.Net  Mobile