title: Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor.
abstract: A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.
authors.affiliation: Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907-1295, USA.
authors.forename: K H
authors.initials: KH
authors.lastname: Hamaker
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: R J
authors.initials: RJ
authors.lastname: Seely
authors.forename: C M
authors.initials: CM
authors.lastname: Ladisch
authors.forename: M R
authors.initials: MR
authors.lastname: Ladisch
mesh_terms: D002848:Chromatography, DEAE-Cellulose; D017510:Protein Folding; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*
chemical_list: D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors