Study On The 50L Fermentation And Efficient Purification Of Recombinant Augmenter Of Liver Regeneration (rALR)

The Augmenter of Liver Regeneration(ALR)plays an important role in liver injury and liver regeneration,and it is a potential targeting drug for liver disease.At present,the domestic and foreign research is mainly aimed at its mechanism of action.Using the biological engineering technology to prepare the recombinant Augmenter of Liver Regeneration(rALR)still stays in the laboratory scale,which can not be large-scale application.In this paper,the key techniques of high expression,big-scale amplification and purification of rALR were studied,and two-aqueous phase extraction technology was introduced for the first time in the purification process,and rALR with high expression,high purity and high activity was obtained.The main contents included:(1)The construction and screening of Pichia pastoris about the highly efficient rALR: first,ALR gene sequence was optimized,then three different kinds of ALR Pichia yeast expression vector were constructed and transferred to three kinds of host bacteria(X-33,GS115 and SMD1168)respectively.The7 kinds of vector-host combinations obtained were identified and induced to express.Finally the high expression the strain of the rALR was screened.The results showed that the expression level of rALR in pPICZ?-A/X-33 was higher than that of pPIC9/GS115,pPIC9K/GS115,and p PICZ?-A/GS115,which was significantly higher than that of pPIC9/SMD1168,p PIC9K/SMD1168,pPICZ?-A/SMD1168.(2)Optimization of expression conditions and system amplification of rALR: In this study,the key factors were optimized and the system was amplified in the expression process,including pH,induction temperature,methanol concentration and dissolved oxygen.The results showed that the best expression pH was 5.5,the optimum induction temperature was 26 ?,the optimum concentration of methanol was 0.25%and when the dissolved oxygen content was 20%,the rONC expression level was highest after 6 days,and the scale of fermentation was successfully enlarged to 50 L pilot scale.(3)The separation and purification conditions of rALR were explored and the purification system was amplified: the rALR 50 L scale fermentation broth was studied,and the condition of isolation andpurification was researched.The results found that the first introduced technique of two-aqueous phase extraction showed using the double-aqueous coupled with ion-exchange chromatography,we obtained rALR with purity rate over 95% and yield rate more than 85%.(4)The biological activity determination of the rALR: the biological activity of rONC was measured in vitro and vivo.At first,The activity of mercapto oxidase of rALR was determined and the effect of rALR on the activity of HepG2 cells was detected by MTT assay.The results showed that rALR had good enzymatic activity and could promote the proliferation of HepG2 belonging to hepatocellular carcinoma cell line.Then function of rALR was determined with CCl4 acute liver failure model and 2/3 liver resection model.The results showed that rALR could obviously promoted and repaired the liver injury and liver regeneration,which mean it had good biological activity.Conclusion: The efficient expression and purification of pilot scale Pichia pastoris were established in this paper.Among them,rALR expression level was 545mg/L in the 50 L scale expression,Compared with the existing literature(424mg / L)increased by 28.5%.Moreover,the corresponding scale of the purification process was established,and the yield was obtained above 85% with more than 95%purity.And the good biological activity of rALR was acquired.which laid a good foundation for elucidating the mechanism clinical research and industrialization of ALR.