Study On Fermentation Of Engineered Escherichia Coli And Purification Of Recombinant MUC1-MBP Fusion Protein

As a member of mucin family,MUC1 is a tumor-associated antigen.Expression of MUC1 on tumor cells and normal cells are quite different,which makes MUC1 become the target of immune cells and also an ideal target for tumor immunotherapy research.In recent years,the research of tumor vaccine targeting MUC1 has received the attention of many scholars.And more and more research has entered clinical trials,what's more,some research is in the stage of phase ? clinical trials now.In 2001,our research group began to take part in this field,WCC1 gene was inserted into pMAL-p2 vector,then the vector was transformed into E.coli to form engineered bacteria which could express MUC1-MBP fusion protein stably and efficiently through the induction of IPTG.With a series of animal experiments,MUC1-MBP fusion protein has shown good effect on tumor inhibition.As a tumor vaccine MUC1-MBP has great development and application prospects.However,the scale of our previous study about engineeried bacteria culture and MUC1-MBP fusion protein purification is too small,which cannot meet the needs of large scale production of MUC1-MBP tumor vaccine.So we need to conduct the research about engineered bacteria fermentation and MUC1-MBP fusion protein purification.This study explores the fermentation of engineered Escherichia coli and the purification of MUC1-MBP fusion protein,and then analyses the quality of MUC1-MBP fusion protein.As a result,we get a large scale of MUC1-MBP fusion protein with high quality,which will lay the foundation for clinical research of MUC1-MBP tumor vaccine.1.Study on fermentation of engineered Escherichia coli expressing MUC1-MBP fusion protein.Firstly,we used four concentrations(0.05,0.1,0.2,0.3 mmol/L)of IPTG to induce engineered bacteria cultured in a erlenmeyer flask in the logarithmic growth phase,then identified MUC1-MBP fusion protein by SDS-PAGE.The results showed that using 0.05-0.3 mmol/L IPTG to induce the expression MUC1-MBP fusion protein were appropriate.Next,with the same concentration of IPTG(0.1 mmol/L)but different induction time(2 h,3 h 4 h,5 h),the expression of MUC1-MBP fusion protein was highest when engineered bacteria was induced by IPTG for 4 h.Therefore,culture conditions in a erlenmeyer flask was optimized.After the bacteria strain was cultured in a test tube and then in a erlenmeyer flask,later inoculated in a 5 L fermenter,we got the time-dependent growth curve after measuring the OD values every one hour.Results showed that logarithmic growth phase of engineered bacteria was 3-12 hours after inoculating them in a 5 L fermenter.Two time points were chosen during logarithmic growth phase(OD values were 10.0 and 15.0)to compare the expression of MUC1-MBP fusion protein,the expression of MUC1-MBP fusion protein was higher when the beginning induction OD value reached 10.0 than 15.0,so 10.0 was chosen as the beginning induction OD value.Then,SDS-PAGE analysis proved that the production of MUC1-MBP fusion protein showed a time-dependent manner,and became stable after 10-12 h.While taking into account that more and more by-products after long fermentation would have a bad effect on the subsequent purification process,so 10-12 h was the optimal induction time.Thus,the fermentation process of engineered bacteria expressing MUC1-MBP fusion protein was initially formed.2.Study on purification of MUC1-MBP fusion proteinSonication method efficiency for crushing engineered bacteria with 30%,32%power had been studied,both 30%power and 32%power had high crushing efficiency.Then we set different time(10 min,15 min,20 min)for sonication process,and found that 10-20 min were suitable sonication time.Crushing efficiency was also detected under optical microscope with crystal violet staining,and most engineered bacteria were broken with 30%power for 10 min.Next,high-pressure homogenization method for crushing engineered bacteria with 1 to 4 times were conducted,SDS-PAGE showed that crushing engineered bacteria with 4 times could release virtually all proteins,and crystal.violet staining indicated that almost all engineered bacteria were broken with 4 times.Considering that 4 times of high-pressure homogenization made the supernatant most clear,which would be beneficial for subsequent purification,so high-pressure homogenization with 4 times was appropriate.For the purification of MUC1-MBP fusion protein,isoelectric precipitation was used to make a crude extract with different pH(3.0,3.2,3.4,3.6)in citric acid/sodium citrate buffer,all these pH caused great loss of MUC1-MBP fusion protein.While with pH 3.4 in NaAc-HAc buffer,the recovery and purity were higher than in citric acid/sodium citrate buffer.So,pH 3.4 and NaAc-HAc buffer were chosen as optimal pH value and buffer.Later,we used cation exchange chromatography to purify MUC1-MBP fusion protein with different concentrations of NaAc-HAc buffer(0.005 mol/L,0.01 mol/L,0.02 mol/L)as the balance solution,results showed that 0.01 mol/L NaAc-HAc buffer could make MUC1-MBP fusion protein firmly combine with the purification medium,while MUC1-MBP fusion protein bound to the medium loosely with the other two concentrations of NaAc-HAc buffer,which would make some loss of MUC1-MBP fusion protein.Next,using eluent with different concentrations(0.3 mol/L NaCl,1 mol/L NaCl)to purify MUC1-MBP fusion protein,we found that 0.3 mol/L NaCl could elute MUC1-MBP fusion protein with higher purity compared with 1 mol/L NaCl.So we chose 0.3 mol/L NaCl as elution concentration.Finally,we successfully obtained MUC1-MBP fusion protein with high purity by using isoelectric precipitation and cation exchange chromatography together.3.Quality analysis of MUC1-MBP fusion proteinHPLC was used to analyse the purity of MUC1-MBP fusion protein,the result showed the purity was beyond 95%;the using of LAL gel assay to detect the residual endotoxin concentration of MUC1-MBP fusion protein indicated the residual endotoxin concentration<5 EU/ml when the concentration of MUC1-MBP fusion protein was 0.5 mg/ml,which was correspond to the specification;epitopes and antigenicity of MUC1-MBP fusion protein were identified by Western blotting.results showed that four concentrations(sample volumes were 1 ?g,2 ?g,4 ?g,8 ?g)of MUC1-MBP fusion protein-had good binding capacity with MUC1 antibody and MBP antibody,which indicated that MUC1-MBP fusion protein had correct epitopes and strong antigenicity.This study preliminarily establishes the fermentation process of engineered Escherichia coli and the purification process of MUC1-MBP fusion protein,which can get large amounts of MUC1-MBP fusion protein with high quality and lay the foundation for the following clinical studies of MUC1-MBP tumor vaccines.