Role Analysis Of Urea And GdnHCl In Protein Folding

Protein folding has always been a difficult problem in the field of life sciences.Nowadays,great progress has been made by the study of nascent peptide chain folding.In vivo,it is clear that the molecular chaperone is the crux to realize the rapid and accurate completion of folding the nascent peptide chain non-folded peptide's hydrophobic surface.The function of it is to recognize the erroneous structures which are temporarily exposed during the folding of the nascent peptide,and to form a complex with the hydrophobic surface from the unfolded or partially folded polypeptide chain.Else to prevent an incorrect non-functional folding from happening,then to inhibit irreversible polymer production.Next to induce the polypeptide chain folding to form the correct conformation and to regulate the energy of the folding process.However,in vitro,most of the eukaryotic proteins expressed by E.coli lack the ability of self-refolding,and spontaneously form an active functional conformation in solution which becomes an insoluble and inactive aggregate called “Inclusion body”.Inclusion body refolding is still a "bottleneck" problem for acquisition large-scale of active functional proteins.In this work,the changes in protein structure,amino acid microenvironment and secondary structure content of lysozyme and carbonic under renaturation of urea and guanidine hydrochloride(GdnHCl)at different concentrations was estimateed using fluorescence spectroscopy,synchronous fluorescence spectroscopy and circular dichroism spectroscopy.Using isothermal titration calorimetry simulated the online dilution renaturation process of Lys/Car and three recombinant proteins(rhDll1,rhG-CSF,STV)and analysis the change of thermodynamic parameters of ?H??S and ?G and main forces of protein folding at different concentrations of denaturants.The thermodynamic equilibrium states of renatured proteins was verified by analyzing the thermal change of the chromatographic fractions of renatured proteins.Recently,the dilution of adding assistants and liquid chromatography have shown the potential in protein refolding by simulating the process and environment of protein folding in vivo.It showed that the addition of urea and guanidine hydrochloride in the refolding buffer or in the chromatographic mobile phase played an important role in the process of protein refolding.Not only can destroy the stability of misfolded intermediates,but also enhance the solubility of folded intermediates and unfolded molecules to increase the yield of refolding.However,the concentration of denaturants which are added to different proteins is quite different from each other.In order to deeply understand the effect of urea and GdnHCl in the process of correct refolding,and to discuss the effects of diverse denaturants in the conformation,microenvironment,the change of main forces and the thermodynamic state of protein folding process.In order to model the protein expression and E.coli inclusions as the research object,in this dissertation,the dilution method and a new hybrid model chromatography are studied,by using fluorescence spectroscopy,synchronous fluorescence spectroscopy,circular dichroism method combined with the isothermal calorimetry(ITC)titration methods.It is distinguished from the spectral properties and the change of the thermodynamic exploration which target in the process of protein refolding,the effect of adding different denaturant and the mechanism of the action.These researches will be of great theoretical significance and guiding significance for scientific research and production in order to find new means which are suitable for efficient renaturational proteins and the production of massive proteins,aim to rapidly promote the industrialization of recombinant proteins and other products.This dissertation is consisted of the following four parts: 1.Literature reviewIn the past decades,great progress has been made by the study of protein folding and the aggregation of pathogenic protein in vivo.It can be said that protein folding is really a basic biological process in nature.Therefore,in order to realize protein folding in vitro,especially in an appropriate environment,such as low concentration denaturants which are aimed to inhibit the aggregations.Proteins which contain disulfide bonds are also eager to have appropriate conditions to form natural proteins.In the part of literature review shows the research and development of protein denaturants and protein refolding,and the progress of main protein folding methods and analysis methods 2.Analysis of the role of Urea and GdnHCl in protein refolding by dilution methodThe study shows that the method of adding dilution assistants plays an important role in protein refolding.Therefore,in order to contain disulfide bond in basic protein lysozyme(Lys)and contains no acidic protein with disulfide bond-carbonic anhydrase(Car),using fluorescence spectroscopy,synchronous fluorescence spectroscopy and circular dichroism method as the analytical means.The purpose is to systematically research the diverse concentration of denaturants(urea and GdnHCl)for the reduction in Lys/Car and summarize the effects,including denaturant concentration on the conformation,folding microenvironment and biological activity.Finally the research found that the guanidine denaturation ability is stronger than urea,and the microenvironment of irreversible process polarity is abate and hydrophobicity.Low concentration of denaturing agents in the refolding buffer is not conducive to the formation of secondary structure of Lys/Car.3.Analysis of refolding effect of denaturant assisted protein by isothermal titration calorimetryThe greatest advantage of the isothermal titration thermal method(ITC)is to provide the thermodynamic parameters in a single titration because of the experimental technique.Therefore,lysozyme(Lys)and carbonic anhydrase(Car)were used as the objects of research.Meanwhile the thermodynamic parameters of on-line dilution renaturation of denatured proteins were simulated by ITC multi-drop experimental system.At 3.0 mol/L,?H < 0,?S < 0,the effect of renaturation on Lys/Car was mainly from hydrogen bond and van der Waals force.However,when the concentration of guanidine was 3.0 mol/L,?H < 0,?S > 0,the refolding effect of Lys was mainly from electrostatic force.When the guanidine concentration at 3.0 mol/L,it showed two stages of Car: the first stage,?H < 0,?S < 0,mainly from hydrogen bond and van der Waals force;the second stage,?H < 0,?S >0,mainly from electrostatic force.The results showed that one-step chromatography could make the conformation of Lys/Car according to the characteristics of natural state and reached the thermodynamic minimum equilibrium state.4.Analysis of denaturant assisted inclusion body renaturation with simultaneous purification by isothermal titration calorimetryOn the basis of the experiment,respectively using the ITC more drops experimental system for restructuring recombinant human Delta-like 1(rhDll1),streptavidin(STV),recombinant human granulocyte colony stimulating factor(rhG-CSF)to carry out the experiments with the on-line dilution method.According to the results from the thermodynamic parameters at the urea concentration of 4.0 mol/L and 5.0 mol/L,?H > 0,?S > 0,the process of 3 kinds of inclusion body which are soluble in strong denaturants showed that in renaturation they are all given priority to hydrophobic force;At the concentration of urea was 3.0 mol/L,STV renaturation process,?H > 0,?S > 0,hydrophobic force was the main force.rhDll1 renaturation process,?H < 0,?S < 0,the effect was mainly from hydrogen bond and van der Waals forces;rhG-CSF renaturation process could be divided into two stages,the first stage ?H < 0,?S < 0,the effect was mainly from hydrogen bond,van der Waals forces;the second stage ?H < 0,?S > 0,the effect was mainly from electrostatic forces.The renaturation and simultaneous purification of STV,rhG-CSF and rhDll1 inclusion bodies under diverse chromatographic modes were analyzed by circular dichroism combined with the method of ITC.