Expression And Purification Of ?B-Crystallin In Pichia Pastoris And The Study Of Its Activity

?B-crystallin belongs to the Small Heat Shock Proteins(HSPs),it has the highest content in lens.?B-crystallin has the activity of molecular chaperones and protect proteins against accumulation,and also will protect cells.?B-crystallin is also a negative regulator,which will prevent the apoptosis of stress cells and retinal photoreceptor cells,and then protect the optic nerve.Recent studies show that the following diseases:age related macular degeneration,glaucoma optic neuropathy,retinal ischemia-reperfusion injury,retinal dystrophy,diabetic retinopathy,retinal tear and nervous system of multiple sclerosis,Parkinson's,Alexander's and Creutzfeldt-Jakob etc,are all associated with ?B-crystallin.In addition,a study of AD shows that there is a relationship between ?B-crystallin and neurofibrillary tangles in the brain of Alzheimer's disease,but the mechanism is not so clear.Therefore,we need to get ?B-crystallin with high purity and activity in order to further study for its structure and physiological function.Because it is difficult to isolate ?B-crystallin from biological purified ?-crystallin completely,and also ?-crystallin is limited in sources and low in yield,which restrict the development and application of a B-crystallin.With the development of protein recombinant technology and the application of recombinant polypeptide drugs in clinical practice,?B-crystallin has been expressed successfully in E.coli expression system and mammalian cells,but it is low in yield and high in price,and renaturation is needed to get active protein.We hope to produce ?B-crystallin by Pichia pastoris expression system,reducing the cost and increasing the yield.This provides a basis for subsequent basic researches and clinical trials of ?B-crystallin and getting recombinant protein with higher activity by changing its structure.In this study,the whole gene was synthesized artificially in vitro and then ligated to plasmid pPIC9K to get CRYAB/pPIC9K.Recombinant plasmids were transformated into Pichia pastoris GS115,after small expression and SDS-PAGE assay,high-expression strains were selected for deeper research.In this study,single assay is used for evaluating the effects of four factors including the fermentation time dosage of methanol,transfer ratio,dosage of methanol and the aeration,in order to get optimal conditions:the fermentation time is 84 hours,the ratio of inoculation is 3:1,dosage of methanol is 0.5%per 12 hours and the aeration is 10/50.Secreted expression of recombinant a B-crystallin were attained by methanol induction and the molecular weight is about 43KD.Recombinant a B-crystallin was purified by Q Sepharose Fast Flow and G-75 Sephadex gel filtration,the yield is 30-40mg per liter of fermentation culture.Insulin reduction assay were used to detect the molecular chaperones activity of recombinant ?B-crystallin,results showed that the recombinant ?B-crystallin could obviously inhibit insulin B chain aggregation deposition reaction caused by the reducing agent-DTT,which proved that the recombinant ?B-crystallin has the molecular chaperones activity.