| Human augmenter of liver regeneration (hALR) is a new hepatic growth factor cloned from liver tissues of human fetal liver. It could increase significantly the survival rate of liver injured rats induced by CCL4. The further studies indicated that it had the biological functions of stimulating the liver cell DNA synthesization,inhibiting the natural killer cell,inducing gene expression of mitochondria and promoting the liver regeneration,what's more,it could oxidate free sulfhydryl and take part in the Fe/S protein synthesis of cytoplasm. The most important function is hALR protection on the acute and chronic liver damage. So the investigation on rhALR would promote the academic research on the liver regeneration mechanism,which would be applied in clinical treatment of severe hepatism.Fermentation condition control of rhALR expressed in E.coli DH5 a,the techniques of inclusion bodies protein denaturation,renaturation and purification,and the biological activities of recombinant protein were investigated in this research with results as follows.l.The fermentative technological conditions of the rhALR expression were determined. It was proved in our optimization screening that the component of optimum medium was as follows:yeast extract 11.7 g,tryptone 5 g,Na2HPO49 g,KH2PO42g,MgSO41.5g,CuSO40.004g,ZnSO4 0.004g,FeSO40.01g,VB10.01 g,D-glucose 10 g,ampicillin 0.01 g,thymine 0.008g,distilled water 1000mL. The fermentative medium was incubated for 5 h under 30 C with pH 7.0 adjusted by ammonia,dissolved oxygen near 30%,then induced for 4 h under 42 C. Glucose and culture medium were added at the rate of 0.25 g/(L h) and 200 mL/h after 3h-incubation,respectively. The wet cell output reached 30 g/L and rhALR expression quantity was 30%.2 The washing procedure of inclusion bodies was improved. A majority of cellular fragment,phospholipid and impurity protein were removed by washing buffer I (50mmol/L Tris-HCl(pH8.0),2mmol/L EDTA,0.2% Triton X-100) II(50mmol/LTris-HCl(pH8.0),2mmol/L EDTA,2mol/L urea),III(50mmol/LTris-HCl(pH8.0),2mmol/L EDTA,70% 2-propanol),IV (50mmol/L Tris-HCl(pH8.0),2mmol/L EDTA,2% sodium deoxycholate)in turn. The inclusion body with 85% purity was harvested.3,The inclusion bodies were dissolved by 6 mol/L guanidine hydrochloride and 50 mmol/L P -mercaptoethanol,then was diluted in the buffer contained 0.5 mol/L Arg to renature for 72 h. The activity experiments verified that the denaturating protein recovered its bioactivity. The renaturing protein was purified by ion-exchange resin (packing:Pharmacia DEAE-Sephadex,chromatography column:XK50/20(CV=392.5mL),flow rate = 10 mL/min,eluant:0-0.5 mol/L NaCl,elute volume=2000 mL),the protein mixture were separated by gel-filtration chromatography (Gel:Pharmacia Sephadex G-50 fine;chromatography column:XK16/80,flow rate = 1 mL/min,eluant:0.9% NaCl) after condensation. Purity of the rhALR protein was 97%.4 The molecular weights of monomer and dimmer based on SDS-PAGE were 15 KD and 30 KD,respectively,and iso-electric point was between 6.55 and 6.85. Analysis of amino acid contents proved that the constitutes of recombinant protein were conformed with the theoretic value. The molecular weight of rhALR was 30088 Da which measured precisely by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS)5 The recombinant hALR protein could increase the survival rate of hepatic failure rats induced by CCL4 and repair the damaged liver cell by mice experiment and stimulate the DNA synthesization in liver cancer cell by H3-Tdr incorporation. It was confirmed that recombinant hALR protein could promote the celluar mitochondria dehydrogenase activity in NIH3T3 by MTT.6 After denatured by urea and then renatured and purified by MALDI -TOF-MS,the molecular weight of rhALR protein was 30780 Da,which was not agree with the theoretic value. Cyanate liberated from urea could reacted with E -amidocyanogen of lysine by measuing the peptide molecular of the enzymatic hydrolysis protein of TPCK-trypsase. The molecular weig...
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