RhG-CSF Mutant Expression And Purification PEG Modification And Biological Characterization

The purpose of this paper is to study the purification and PEG modification process of company patented rmhG-CSF-Cys17,and the biological activities of PEG modified rmhG-CSF-Cys176.The research contents include rmhG-CSF-Cys¹⁷⁶ fermentation liquid separation and purification of high purity is obtained,the rmhG-CSF-Cys¹⁷⁶ protein,and the protein and polyethylene glycol maleic imide group?mPEG-Maleimide,mPEG-Mal?specific binding,research the modification reaction conditions,access to poly two alcohol modified product mixture,and the mixture is separated and purified,high purity PEG-rmhG-C SF-Cys176,and the modified protein on biological characteristics of PEG-rmhG-CSF-Cys¹⁷⁶ scale production,and provide experimental basis and foundation.Research findings:1.mutant rmhG-CSF-Cys¹⁷⁶ preparation and purification and identification of mutants:rmhG-CSF-Cys¹⁷⁶ strains fermentation,solubilization,refolding,hydrophobic interaction chromatography,HIC Resource S ion exchange chromatography is the purity of RP-HPLC 99.2%,SDS-PAGE 99%purity,high performance gel filtration the purity of high purity of 100%rmhG-CSF-Cys¹⁷⁶ protein.2.rmhG-CSF-Cys¹⁷⁶ PEG modification and purification:rmhG-CSF-Cys¹⁷⁶ protein and polyethylene glycol maleic imide group?mPEG-Maleimide,mPEG-Mal?modifying reaction,research has identified PEG modified rmhG-CSF best condition:the 50ml lmg ml-1 rmhG-CSF-Cys¹⁷⁶ 100 mM Bicine?pH 6?,then adding 2 times of the molar ratio of polyethylene glycol maleic imide base?PEG-MAL20000?in 18 ?,reaction under stirring 24h,the conversion rate can reach 43%.Through the Resource S ion exchange chromatography on PEG-rmhG-CSF-Cys¹⁷⁶ modification reaction mixtures separation and purification,purity of 98.9%PEG-rmhG-CSF-Cys¹⁷⁶ protein.3.Study on the biological characteristics of 3.PEG-rmhG-CSF-Cys176:the modified protein on biological characteristics study found:PEG-rmhG-CSF-Cys¹⁷⁶ activity and rhG-CSF activity is higher than that of PEG-rhG-CSF?N?,active;activity in vivo experiment was long lasting,animal experiments confirmed,its in vivo half-life is prolonged to 14.8 hours,than unmodified rhG-CSF?T1/2=2.2 hours?is improved by 7 times,reached long-term objective.