| Protein renaturation is a recognized bottle-neck technique in the down-stream in biotechnology.The key point here is how to make the stable intermediates(M state) of protein fold into its correctly state.Protein folding liquid chromatography(PFLC) is a recently developed new method for protein renaturation.It is not only employed for carrying out the separation of protein in different states,but is also for the investigating the changes in molecular conformation of proteins.With some linear parameters of stoichiometric displacement theory(SDT),the different molecular conformations of proteins can be characterized.Thus,using the PFLC and SDT to study on the proteins folding and the folded intermediates is a new approach in biotechnology and molecular biology.Two-dimensional liquid chromatography (2DLC)and multi-dimensional liquid chromatography(mDLC)have become very strong tools for the separation and analysis of some components in a complex system in analytical chemistry,especially for the pre-fractionation of intact proteins in proteomies.Due to some shortages of the present separation of proteins by 2DLC or mDLC,such as high cost,long separation process,unsatisfactory reproducibility,their applications are greatly limited.Therefore,the method for improving 2DLC,or mDLC has become one of researching hot-point in both proteomics and LC in the world.The thesis contains five parts as follows:1.Literature review:The intermediate(M state)formed during protein folding retards its corresponding denatured protein(U state)to convert to its native state (N state).Because the lifetime of the M state is so short that it is very difficult to be captured and separated,the study on the folded M state has a greatly theoretical signification and widely applied value.This research in this field still places at an experimental stage.The formation,researching strategy,method and recent development of the M state during protein folding were comprehensively reviewed in this chapter.Some unsolved difficult problems,and in the future of 2DLC mDLC for the separation of proteins in N state were also introduced and commented.The thesis contains 160 references.2.The M state of urea-denaturedα-chymotrypsin(α-Chy)was selected as a model M state and its character was investigated by PFLC.By using hydrophobic interaction chromatography(HIC),a stable M state ofα-Chy was prepared and separated on-line under a dynamically chromatographic condition.The three linear parameters of Z(A constant corresponding to moles of water is squeezed out at the contact region between protein and stationary phase as one mole protein is adsorbed by stationary phase),lgI(a batch of constant corresponding to the affinity of one mole protein to stationary phase),and j(a batch of constant corresponding to the affinity of one mole water to stationary phase)in SDT ofα-Chy in M and N states were determined under different concentrations of urea. The obtained result demonstrates that the three parameters of theα-Chy in M and N states are quite different with each other,a very good linearity relation(Slope is j)was found to exist between the Z and lg I of theα-Chy in N state,but not for its M state.It was also found that the value of Z and lgI of theα-Chy in M state is less than its corresponding N state.Then the different molecular conformations of theα-Chy can be distinguished by the different of the three parameters,and established a new method for the characterization ofα-Chy intermediate by online PFLC3.The retention of the obtained M state ofα-Chy during protein folding by six different kinds of HIC stationary phases was compared with each other.The surfaces of the HIC stationary phase of PEG-600 and TSK-phenyl were selected as the two typical solid surfaces to study the folding efficiency of the urea-denaturedα-Chy andα-Chy in M state.The obtained result indicates that the character of a solid surface has a significant contribution to protein folding. Compared with the TSK column,the surface of PEG-600 stationary phase is efficient for the renaturation of the urea-denaturedα-Chy and for its quality control duringα-Chy folding.Matrix-assisted laser desorption ionization time of a flight mass spectrometer(MALTI-TOF MS)was employed to measure the molecular mass of each collections after HPHIC separation by the two kinds of stationary phases and to confirm that there is only one stable folded intermediate from the mixture of the urea-denaturedα-Chy.With the comparison of specific bioactivity of the refoldedα-Chy,the surface of HIC PEG-600 stationary phase was proved to be better than that of TSK again.It further demonstrates that a suitable hydrophobic surface with a suitable hydrophobic strength and a ligand structure plays a key role in protein folding.4.Three kinds of specific stationary phase having the character of both weak cation-exchange chromatography(WCX)and HIC,respectively,were synthesized. It was proved by experiment that these stationary phases have very good resolution for proteins separation,ether in ion-exchange chromatography(IEC) mode,or HIC mode,and this column is expressed as WCX～HIC column.The principle of protein separation with the WCX～HIC column was briefly described. The character of protein retention on the stationary of the WCX～HIC column can be controlled by the kinds of ligands,the amount of added reagent amount and the differently synthesized condition.It was found that this WCX～HIC column has a great dynamic column capacity under the two chromatographic modes, respectively.Furthermore,the mass recovery of several proteins under the two modes was found to be higher than 96%,this result indicates that the in-reversible adsorption on this stationary phase may be ignored.Based on all of the foregoing results,a new method that with only a single chromatographic column to accomplish the separation of intact proteins by the combination of WCX and HIC is firstly presented.With selecting a suitable stationary phase,mobile phase, buffer exchange condition,and sample injection by two times,the protein separation with usually 2DLC mode can now be accomplished using the single column only in one hour.By compared with the very good commercial TSK WCX and HIC columns,respectively,the resolution of the WCX～HIC column can be comparable with the two TSK columns.Due to the WCX～HIC column can be used in 2DLC with the column capacity(n1×n2),the peak capacity of the WCW～HIC column is much greater than one normal column.5.When the synthesized single WCX～HIC column is employed for a 2DLC separation of proteins,it is called as the two-dimensional liquid chromatography by a single column and expressed by 2DLC-1C.The 2DLC-1C can be employed by either off-line,or on-line for protein separation.The latter is called as on-line 2DLC-1C.With solving the on-line buffer exchange,sample and/or collection of large volume(0.001-100mL)on-line injection by either,continuously,or discontinuously,the 2DLC-1C is firstly used for on-line 2DLC for protein separation in both analytical and preparative scales.Moreover,the 2DLC-1C was applied for standard proteins and blood serum rapid separation;the obtained results were proved to be excellent.The advantages of the 2DLC-1C are that all of operations are:(1),Whole protein separations are carried out in a closed system, prevent from any contaminations;(2),All of components in original sample are quantitatively transferred to the subsequent operation;(3),Each protein remains native state;(4),The complexity of the original sample is simplified very much, such as from 1/10～1/100,even much less;(5),The relative enrichment of each proteins,especially for very low enrichment proteins in proteomics can be increased up to 10～100 folds,even much more;(6),It is easily for operation atomization;(7),fast Native and/or intact protein can be fast separated by means of on-line matter;(8),It can not only obtain reliable information,but also obtain pure proteins in proteomics;(9),A high mass and bioactivity recovery for isolation from natural product,or animal fluids can be obtained;(10),Its expense is very low.
|
PDF Full Text Request