High-Level Expression And Purification Of Recombinant Staphylokinase

Thrombus is the major leading life-threatening disease human faced, including acute myocardial infarction,pulmonic infarction,artery infarction,shock lacked of blood and so on. The mortality is more than 30% caused by acute myocardial infarction. Thrombolytic treatment is considered as the best way to cure thrombus,it is always a hot point to study better thrombolytic drugs with fewer side-effect in the world. It has been demonstrated that Recombinant staphylokinase (rSak) induces fibrinolysis specifically without fibrinogen depletion and has higher fibrinolytic activity compared with other plasminogen activators such as SK, urokinase and tPA. In addition,rSak has been shown to be more efficient than SK for the dissolution of platelet-enriched and retracted blood clots. Therefore in recent years,rSak has become a promising drug and stimulated much structural and protein engineering research.In this report,rSak was expressed in a soluble and active form. This study comprehensive optimize the fermentation of recombinant E.coli expressing rSak,and downstream process of rSak was investigated.Moreover,the results were helpful to be scaled up to the industrial production of rSak.The main contents include:I. Study on the optimizing of fermentation process.Il.Study on the purification and identification of rSak. III. Analyze the activity of rSak.The main results of our study include:I,The recombinant E.coli strain was incubated in the flasks and the optimum conditions for cell growth and expression of rSak were as follows:glucose,yeast extract:tryptone(1:2),temperature 30-32℃.II,The given fermentation parameter was applied to Biostar 70 litre fermentation tank in a pilot scale. It was found that induction time affected cell growth and expression of rSak,on the same time,the optimum induction time was 3-4h.III,The pilot-scale fermentations were thoroughly studied by orthogonalexperiment design. During the fermentation process,the conditions of some expression factors,such as pH,DO saturation and supplement,et al,affected rSak expression. The results were as follows: glucose 2%,nitrogen resource 1.5%,pH6.8,DO saturation30%.Under these conditions,the average expression of rSak was 52%,A₆₀₀ was 35.8 and DCW was 8.2g/L.IV,Ultrafiltration was used to concentrate and desalt solutes retained by the membrane or to collect material passing through the membrane. Purification process was established starting with precipitation of 30%(NH₄)₂SO₄,Ultrafiltration, followed by ion-exchange chromatographies on DEAE-Sepharose FF and SP-Sepharose FF.Under these optimum conditions, protein yield was 32% and the purity analyzed by SDS-PAGE was over 99%.V,The production was repeatedly obtained with the stable expression, protein yield and purity of rSak.The purified rSak migrated as a single protein band on reduced/non-reduced SDS-PAGE was above 99%.Molecular weight of single peptide chain at reduced SDS-PAGE condition demonstrated 15.5 kD.By inspections of SDS-PAGE,HPLC,UV absorption spectrum,residual antibiotics and bacterial endotoxin,the sources of rSak from continuous three batch were up to the standard of biologics.VI,The activity of rSak was measured by using the fibrin plate method that was more rapid and sensitive than others. The respective results of activity were as follows:2.2×10⁸,3.1×10⁸,2.8×10⁸IU/ml.It was confirmed that the whole fermentation and purification process were suitable and stable during rSak production.