Pilot Fermentation And Purification Of Recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8)

LZ-8 is a Fungal Immunolomodulatory Protein from the mycelium residues of Ganoderma lucidum in 1989 by Kino,it has high performance in immunoregulatory function.For the present,LZ-8's known activities mostly are about agglutinating sheep red cell but not human's,it can also prevent the allergic reactions of rats as well as the antibody production.Our study team have recombinant expressed LZ-8 in P.pastoris for the first time,while we already have established the technology of fermentation and purification in pilot scale,and its immuno-activities was identified and examined by testing macrophages phagocytosis of mouse and agglutinating of sheep red cell.The study on the large-scale fermentation technology of rLZ-8Making full use of advantages of Pichia pastoris expression system,this study carried out the process of large-scale pilot scale production of rLZ-8 and then optimized the parameters of technique.Pichia pastoris has a wide growing pH range from 3.0～7.0.Cells in the growth phase can produce acid in order to reduce pH value of environment gradually;In addition,the cells can release proteolytic enzymes,mainly is basic hydrolase;And large-scale fermentation by continuous supply of ammonia to maintain the pH,and buffer system in the culture medium of our study consisted inorganic salts,too high pH values leads to the precipitation.On the basis of results of flask experiment,pH=5 parameter was chosen for optimizing amount of rLZ-8 in the large scale fermentation.In the fermentor,the culture stage,cell mainly depends on carbon source of BMGY medium to enhance the quality of Bacillus.Then we Initiate a 50%w/v glycerol feed containing 12 mL PTM 1 trace salts per liter of glycerol feed.Set the feed rate to 15 mL/hr/liter initial fermentation volume and keep DO value more than 30%.All of the glycerol needs to be consumed and keep 'hunger' for 30 minutes before starting the methanol feed to fully induce the AOX1 promoter on methanol.When wet weight increase 220～240g/L,the process of fermentation enters methanol fed-batch phase,also named 'induce phase'.Parameters setting: 0-2mL/h/L(0-2h) 2-5mL/h/L(3-8h),5-10mL/h/L(9-16h),10mL/h/L(17-47h),10-2mL/h/L (48-58h),2mL/h/L(59-103h);when fermentation enters 48-58 hrs period,decreases or increases the amount of methanol addition according to the strain weight in humidity of more or less than 350g/L,decrease or increase the flow of additional methanol;when fermentation enters the late 70 hrs,namely the so-called induction period,add at 1mL/h/L rate of a 0.5M (NH₄)₂ SO₄ 10h.One thing which is equally important to methanol is control strategy of DO.In all steps of fermentation,these strategies are as follows:1.When DO＞50%,main causes are 'Less carbon sources in fermentation broth 'and 'High access to oxygen,but this increased DO value doesn't keep long,it is solved with the increase of biomass in the fermentor',main solutions are 'Supplement of glycerol or methanol' and 'Adjust various parameters of the air mixer';2. When 20%＜DO＜50%,main causes are 'Dissolved oxygen value maintains such a value,it is relatively stable',main solution is 'Adjust all parameters every 6 hours each time to keep the numerical';3.when DO＜20%,main causes are 'Faster supplement of glycerol or methanol.Low access to oxygen,or it may be not enough rotation rate,that makes it inadequate to dissolve more oxygen in fermentation broth',main solutions are 'If by supplementing oxygen and speeding-up to keep the motor speed that the dissolved oxygen is stable at a reasonable value,low supplement of carbon source is not encouraged.'.The 40L fermentor was used in the study of rLZ-8 expression in Pichia pastoris for the large-scale fermentation.The study focused on a greater contribution on pH,compositions of pilot culture medium,dissolved oxygen(DO value),add speed of methanol,the initial biomass and some other factors.All experimental data exhibited the parameters in the process of fermentation as follows:0.5%peptone in medium,pH5.0,wet weight 200g/L before inducing, DO value which is between 20%and 30%is more appropriate,yield:800mg·L^-1.2.Establishment of rLZ-8 purification technologyThis experiment also explored the large-scale purification methods of rLZ-8.Put 30L centrifugal supernatant of fermentation broth through 10k hollow Fiber Ultrafilter,and then through 3K Hollow Fiber Ultrafilter to concentrate and to eliminate the.salt.Delute the 6L sample with Tris-HCl buffer(20 mM,pH 7.5) to 18L.Purify through Q Sepharose XL anion exchange resin,6 L regurgitant liquid was obtained,then through 3K Hollow Fiber Ultrafilter to concentrate and to eliminate the salt.3.the purification process route of rLZ-8Firstly,put the sample through ion-exchange chromatography using the column of Q sepharose HP,then trough hydrophobic chromatography.Finally,through gel chromatography. Determine the purity and molecular weight of the collected rLZ-8.The result showed that purity of rLZ-8 is＞95%,molecular weight is 12,722Da.4.Immunocompetence identification ofRlz-8 immunodulatory proteinThe immunological activity of Rlz-8 immunodulatory protein was proved by a series of experiments as Macrophage's Swallowing Chicken Red Blood Cells in Mouse's Abdominal Cavity,the influence of rLZ-8 protein on human blood types and sheep red blood cell agglomeration.To sum up,we investigated and optimized the qualification of ferment and purification for rLZ-8 Pichia pastoris engineering bacteria in 40L fermentor.It would lie a theoretical foundation for rLZ-8 industrial production and further clinical application by our fine purification and immunocompetence identification.