Abstract: To establish an industrial method of recombinant neorudin(EH) expressed in Escherichia coli,low density fermentation and high density fermentation were performed in this study,and in the high density fermentation,the fermentation culture medium and the induction time were investigated.Sonication and repeated freezing and thawing method were carried out to evaluate the release of EH from E.coli.SP Sepharose Fast Flow cation exchange chromatography and Source 15 Q antion exchange chromatography were chosen to purify EH.The purified protein was identified by Western blot,its purity was analyzed by HPLC,and the special anticoagulant activity was detected by a clot method.Finally the purified protein was confirmed with the high resolution mass spectrum,C-terminal amino acid sequence analysis,N-terminal amino acid sequence analysis and disulfide bond analysis.The result shows that the high density fermentation based on TB culture medium is established.OD₆₀₀ was about 40 after fermentation,the wet weight of cell was about 70 g/L,and the expression level of EH was about 400 mg/L.Compared with sonication,repeated freezing and thawing method ensured EH release with less irrelevant proteins.After a two-step ion-exchange chromatography,the purity of EH analyzed by HPLC was 97.84%.The special anticoagulant activity of EH protein was about 1 024 ATU/mg after incubation with bovine coagulation factor Xa.Furthermore,the molecular weight of EH was determined to be7 415.1880 ku by high resolution mass spectrum.The results of C-terminal amino acid sequence analysis,N-terminal amino acid sequence analysis and disulfide bond analysis show that the EH protein produced with the production methods is right.A fermentation and purification method of EH expressed in E.Coli is developed.