Prokaryotic Expression And Renaturation Of Streptavidin

Streptavidin (SA) is a biotin binding protein naturally produced from Streptomyces avidinii via secretion and exceptionally high affinity to water-soluble vitamin,D-biotin. SA-biotin system has advantage of high affinity, sensitivity, specificity, tability and magnification for signal. The SA-biotin system has been widely used for many applications, such as immunology, medicine, molecular biology, histochemistry. The resource of the SA is come from the production of its natural host or recombinant expression from E. coli or S. subtillis. The last onne takes advantadge of shrot production cycle, high yield and easy purification, and the reaserch of recombinant streptavidins is a focus these days.The E. coli and S. subtillis expression system are widely used for recombinant proteins now. E. coli and S. subtillis system expression has advantage of overexpression, wide application and clear background of genetics, and the S. subtillis expression system has advantage of secretory express and safety for production.In this research, aim to obtain the over-expression strain and the renature plans of the recombinan tSA, some different styles SA were expressed by S. subtillis expression and E. coli expression system and renatured in this research.Three different DNA fragments, stv-13(encoding 16-133AA), csa(encoding 13-139AA), cnsa(encoding 13-159AA) were amplified by PCR from Streptomyces. avidinii genomic DNA. The stv-13 was subclonied to pP43, pSacR expression vectors and constructed three expreesion strains: B. subtilis WB800(pP43-stv-13), B. subtilis168(pP43-stv-13), B. subtilisWB800(pSacR-stv-13). The stv-13, csa and cnsa were subcloniced in pET22b, pET11a, and constructed E.coli expression strains: BL21(DE3)(pET22b-stv-13), BL21(DE3)(pET22b-csa), BL21(DE3)(pET11a-cnsa), BL21(DE3)pLysS(pET22b-stv-13), BL21(DE3)pLysS(pET22b-csa), BL21(DE3) pLysS(pET11a-cnsa), ER2566(pET22b-stv-13), ER2566(pET22b-csa), ER2566 (pET22b-cnsa). After induced by IPTG, the recombinant protein was overexpressed by BL21(DE3) pLysS(pET11a- cnsa). The SA was obtained more than 40% quantity of whole cell protein and the renature efficiency is 80% after optimizing the condition of expression and renature. ELISA analysis show that SA have some bioactivity as standard SA(Promega).