Optimization for Purification and Characterization of Recombinant Hirudin III from E. coli

Abstract

Abstract: Aim To optimize purification conditions of recombinant hirudin 3 in the fermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated and purified from the fermentation broth by three column chromatography steps with macroporous resin, DEAE cellulose DE52 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 was determined by mass spectrometry. The structure of the product was analyzed by peptide map. Results The product with purity of 95.4786% was obtained after three purification steps in the optimum conditions 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 was coincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfully employed for purification of r-hirudin 3 from E. coli using batch-type approaches. The product obtained with high purity was confirmed to be r-hirudin 3.

Key words: recombinant hirudin 3, recombinant hirudin 3, purification, purification, macroporous resin, macroporous resin, RP-HPLC, RP-HPLC, mass spectrometry, mass spectrometry, peptide map, peptide map

CLC Number:

R917.796

R996.3

Supporting:

WEI Li-jun, LIU Jun, WU Bin, LI Xue-feng, YE Shuang-ning, ZHANG Liang, WU Wu-tong^*. Optimization for Purification and Characterization of Recombinant Hirudin III from E. coli[J]. .

References

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