The Renaturation And Purification Of Human Ribonuclease Inhibitor

Human Ribonuclease Inhibitor( hRI) is an acidic 50 kD protein which is located in the cytoplasm. hRI consists of 15 leucine-rich repeat(LRR).There are 32 highly conserved cysteine residues which must be reduced because it is necessary for hRI to maintain biological activities. This feature allow hRI to function only in a reducing environment such as cytosol. Tertiary structure of hRI is symmetry horseshoe-shape. RI binds pancreatic type ribonucleases with 1:1 stoichiometry and competitively inhibits their ribonucleolytic activity. RI can also bind with Angiogenin, inhibiting its angiogenic activity. hRI is located in the number 11 chromosome (11p15.5). There are often aberrant in the 11p15.5 among cancer patients. So RI may be involved in proliferation and differentiation of cell. As we all know, Neovascularization is necessary for tumor growth and metastasis. It is one of methods for tumor therapy to prevent the tumor growth and metastasis by inhibiting the neovascularization. Angiogenin is present in the both normal tissues and cells and cancer tissues and cells. Angiogenin targets on endothelia of the blood vessel, promotes the endothelia proliferate and demonstrates an intense angiogenic activity. Interaction between hRI and angiogenin can make hRI become a potential medicine for cancer therapy. The animal experiments also demonstrated that RI can inhibit the transplant tumor cell growth of mice including Ca761,S180,H22 tumor in the animal body. Our experiments also showed that RI activity decreased in the blood of tumor patients and RI mRNA was significantly lower in the breast cancer tissues than in the normal tissue.RI can preferably apply to cancer therapy and RI extracted from tissues is low yields, high cost which isn't fit for a batch production. Therefore methods which produce RI by gene recombination have being researched by our laboratory. But when a recombination human ribonuclease inhibitor was expressed in E.coli, the expression products accumulated as inclusion bodies. Inclusion bodies are aggregation which is formed during over-expression of recombination proteins. Such recombination proteins don't have biological activities. This problem made us be caught in the dilemma. So this paper mostly research renaturation and purification of recombination hRI. Our work include:1.Expression of hRI in the E.coli: Extraction recombination plasmids DNA pET23b-ri by Alkaline Lysis; transformation of competent E. coli BL21(DE3) using calcium chloride. Put E.coli BL21(DE3) into LB medium containing ampicillin and make them propagate until A600=0.6, then add inducer IPTG to induce hRI express.2.Purification and solution of inclusion bodies: Lysis cells by sonication and centrifuge the cell lysate .Retain pellets which are inclusion bodies. Wash pellets by buffer containing detergent again and again in order to get rid of the lipids, membrane proteins and proteins adsorbed onto the inclusion body. At last dissolve pellets with buffer containing 8M urea and 20mmol/L β-mercaptoethanol .3.Renaturation of inclusion bodies: The renaturation of hRI was performed by three methods: dilution,gel filtration,dialysis.1)Dilution: add non-ionic detergent TritonX-100 into refolding buffer in the process of dilution in order to promote hRI refold. Refolding conditions including concentration of additive TritonX-100,initial concentration of unfolding proteins,refolding time were explored . 2)Gel filtration: the hRI was refolded on a Sephadex G-150 (10mm×300mm), concentration of sample proteins was about 1mg/L, sample volume was 0.4-0.8ml, flow rate was 4—6ml per hour.3)Dialysis: the urea concentration of dialysis buffer has been decreased linearly from 4mol with a rate of 0.3mol/h for five hours. Maintained the urea concentration of dialysis buffer which was about 2.5mol/L and continued to dialyze for twelve hours, then restarted to reduce linearly the urea concentration of dialysis buffer for seven hours. 4.Affinity chromatography: ligand is RNaseA. Affinity chromatography column CNBr-a...