Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column...Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.展开更多
More and more concerns about health bring the increasing demand for blood contact tissue engineering alternatives.In this paper,nanoparticles of poly(lactic-co-glycolic acid)/polyethyleneimine mixed with recombinant h...More and more concerns about health bring the increasing demand for blood contact tissue engineering alternatives.In this paper,nanoparticles of poly(lactic-co-glycolic acid)/polyethyleneimine mixed with recombinant hirudin(rHNPs)were prepared by a double emulsion solvent volatilization method,which were then loaded onto the polycaprolactone(PCL)with polydopamine(PDA)coating to form the composite nanofibers of PCL/PDA/rHNPs.The hydrophilicity and mechanical properties of the composite nanofibers were improved significantly compared with pure PCL.The morphology kept almost unchanged after 30 d of degradation in phosphate buffer saline(PBS).The anticoagulant molecule of hirudin could be gradually released from the composite scaffolds through the degradation of rHNPs in vitro.When the concentration of rHNPs suspension was 5.0 mg/mL,the composite nanofibers could better promote the growth and proliferation of human umbilical vein endothelial cells(HUVECs).The anticoagulant ability of the composite nanofibers was also significantly improved in comparison with that of pure PCL.The design of controlled release anticoagulant materials would alleviate the sudden release of simple fixed hirudin,which could also provide a new idea for the development of novel blood contact materials.展开更多
文摘Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.
基金supported by the Key Technologies Research and Development Program of China(No.2017YFC1105000)the Natural Science Foundation of Guangdong Province of China(No.2018A030310374)+2 种基金the Guangdong Medical Research Foundation of China(No.2022YDZ09)the Fund of Guangzhou Science,Technology and Innovation Commission of China(No.202102080430)the Fund of Higher Education Discipline Innovation Project of China(No.B13039).
文摘More and more concerns about health bring the increasing demand for blood contact tissue engineering alternatives.In this paper,nanoparticles of poly(lactic-co-glycolic acid)/polyethyleneimine mixed with recombinant hirudin(rHNPs)were prepared by a double emulsion solvent volatilization method,which were then loaded onto the polycaprolactone(PCL)with polydopamine(PDA)coating to form the composite nanofibers of PCL/PDA/rHNPs.The hydrophilicity and mechanical properties of the composite nanofibers were improved significantly compared with pure PCL.The morphology kept almost unchanged after 30 d of degradation in phosphate buffer saline(PBS).The anticoagulant molecule of hirudin could be gradually released from the composite scaffolds through the degradation of rHNPs in vitro.When the concentration of rHNPs suspension was 5.0 mg/mL,the composite nanofibers could better promote the growth and proliferation of human umbilical vein endothelial cells(HUVECs).The anticoagulant ability of the composite nanofibers was also significantly improved in comparison with that of pure PCL.The design of controlled release anticoagulant materials would alleviate the sudden release of simple fixed hirudin,which could also provide a new idea for the development of novel blood contact materials.
