Study On Refolding With Simultaneous Purification Of The Expressed Notch Ligand Delta-like1 In Escherichia Coli

It has been a bottleneck for the preparation of high activity and quality recombinant protein drugs.At present,high performance liquid chromatography(HPLC)which has a good performance on separation and rapid purification of proteins,has been an important method for rapid purification and refolding of the inclusion body.PFLC has the advantage of the good separation effect,the strong applicability and the large-scale production of recombinant protein drugs,while the IEC/HIC(hydrophobic interaction chromatography(HIC)/ion exchange chromatography(IEC))orthogonal mixed mode chromatography(MMC)plays an important role in improving the efficiency of protein fold'ing.In preliminary work,four kinds of MMC(IEC/HIC)stationary phase with cholic acid,phenylethylamine,3-phenyl-propylamine and tryptophan as ligands was prepared respectively,which successfully achieve the refolding and purification of lysozyme and rhDlll-RGD expressed in E.coli and this four kinds of chromatographic stationary phase all display5 eharacteristics that combine both the hydrophobie interaction as the main interaction and the electrostatic interaction as the assisted one.It was found that the PFLC stationary phase with selective electrostatic force has a promoting effect on protein refolding in HIC mode.Therefore,in order to study the effects of this four kinds of chromatographic stationary phase on the refolding and purification of proteins,in this paper,rhDlll was expressed in E.coli firstly,and the fermentation conditions were optimized and the rhDll1 was crude separated.The difference between the rhDll1 used in this paper and the rhDlll in previous studies is that the short fused peptides such as histidine tags and RGD oriented peptide tags are removed and the fusion tags are no need to be cut.However,rhDll1 mainly exists in the form of insoluble inclusion bodies,so the obtained rhDlll cell bodies needed to subjected to ultrasonic cell disruption and washed by buffer solution,and the inclusion body needed to be dissolved in the solution of 8 mol/L urea.On the basis of previous studies,four stationary phases with cholie acid,ethylamine,amphetamine and tryptophan as ligands were synthesized and characterized,and four kinds of chromatographic columns were filled by this four stationary phases.The study of renaturation and purification of rhDlll inclusion bodies with different ligands at different scales provides reliable data for the solution of the complexity of inclusion bodies.1.Literature reviewThe research involved in the field of life science,separation science and other related fields.Therefore,this section focuses on the most important signaling pathway for the regulation of cell proliferation and differentiation(including Notch signaling pathway,Notch ligand and receptor protein),the characteristics of efficient expression of recombinant protein in E.coli(including soluble and insoluble recombinant protein)and the protein folding liquid chromatography(Protein folding liquid chromatography,PFLC the literature review method).2.The high expression of recombinant human Delta-like 1 protein in E.coli and the crude separation of its inclusion bodyThe influence of fermentation conditions of temperature,inducing time and rotation speed on the expression the target protein and growth of E.coli was investigated.The results showed that 4%of the E.coli was added to the medium LB,which were cultured in 37?and shaked at 220 r/min,and added the IPTG at logarithrnic growth phase after 4 hours the expression of protein can reach maximum,and the highest expression level was 40.5%.The crude purification process of inclusion body was analyzed.The results showed that with the increase of washing times,the purity of protein increased gradually,and the total protein recovery rate decreased.Finally,the inclusion body was dissolved in the solution of 8 mol/L urea,and the content of the protein in the solution was 5.6 mg/mL,while the purity of the protein was up to 55.6%.3.Refolding and purification of recombinant human Delta-like 1 protein by different columns at hydrophobic interaction chromatography modeFour kinds of chromatographic stationary phases were prepared by ourselves to achieve the refolding and purification of rhDlll protein.First,the four kinds of MMC stationary phases with cholic acid,ethylamine,amphetamine and tryptophan as ligand were synthesized,and four kinds of chromatographic columns were filled.Then,the effects of the chromatographic conditions,including the elution gradient,the inject volume,the pH,urea concentration and salt concentration of the mobile phase on the refolding and purification of rhDlll were investigated.The result showed that the denatured protein on these four kinds of MMC stationary phase follow the mechanism of both the hydrophobic interaction as the main interaction and the electrostatic interaction as the assisted one.The electrostatic force on the HPHIC column has a regulatory effect on the refolding of proteins,and the MMC stationary phases with several forces are also favorable for protein refolding.Under the optimal chromatographic conditions,the mass recoveries were:tryptophan(69.3%)>phenylethylamine(62.8%)>cholic acid(59.3%)>3-phenyl-propylamine:59.2%,and the purity were:phenylethylamine(95.8%)>cholic acid(94.5%)>3-phenyl-propylamine(93.3%)>tryptophan(93.2%).Fluorescence spectra and circular dichroism spectra were used to characterize the refolded proteins.The results of the two spectras showed that rhDlll was successfully refolded.4.Refolding and purification of recombinant human Delta-like 1 protein by hydrophobic interaction chromatography cakes at chromatography.On the basis of the optimization of the chromatographic column,the rhDlll refolding and purification were carried out by using a kind of large-scale column,chromatographic cake.According to optimization of the flow rate and injetion volume of the sample,the results showed under the optimal chromatographic conditions,the mass recoveries were:tryptophan(62.2%)>phenylethylamine(59.8%)>cholicacid(55.8%)>3-phenyl-propylamine(53.2%),and the purity were:cholic acid(95.2%)>tryptophan(94.9%)>phenylethylamine(93.7%)>3-phenyl-propylamine(93.2%).Protein purity and molecular weight were analyzed by SDS-15%PAGE and mass spectrometry(MALDI-TOF-MS),Fluorescence spectra and circular dichroism spectra were used to characterize the refolded proteins.The results of the two experiments showed that the rhDlll was successfully refolded on these four chromatographic cakes.