title: [Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds].
abstract: To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
authors.affiliation: West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
authors.forename: Weiquan
authors.initials: W
authors.lastname: Xie
authors.forename: Guifeng
authors.initials: G
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Gao
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.forename: Rong
authors.initials: R
authors.lastname: Yu
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D005343:Fibrinolytic Agents / Q000032:analysis / Q000737:chemistry; D006629:Hirudins / Q000032:analysis / Q000737:chemistry*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000737:chemistry*
chemical_list: D004220:Disulfides; D005343:Fibrinolytic Agents; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C087896:reteplase; D010959:Tissue Plasminogen Activator