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.

Rapid chromatography for evaluating adsorption characteristics of cellulase binding domain mimetics

“…Cotton and cotton/polyester blend textiles in tightly rolled cylinders have been shown to make good continuous stationary-phase materials for rapid chromatography (Hamaker et al, 1996(Hamaker et al, , 1998Li et al, 2002). The material chosen for the adsorption chromatography screening was an untreated cotton print cloth, which is nearly 100% cellulose.…”

“…One commonality between these various compounds is a negative charge at pH 2.2. Cellulose has a native cation-exchange capability (Hamaker et al, 1996), thus the solute exclusion is most likely due to the negative charge on the cellulose surface repelling these compounds from the pore volume.…”

Refolding of Mis-folded Recombinant Cytochrome c 3 with Strong Cation Exchange Chromatography

“…[11] Of these methods, liquid chromatography has been paid more attention due to its preparative ability in industry. Especially, ion exchange chromatography has been successively employed for the refolding of many proteins, such as papiloma virus HPV E7MS2, [12] fusion proteins of monomeric a-glucosidase, [13] recombinant secretory leukocyte inhibitor, [14] lysozyme, [15] and bovine serum albumin. [16] In the studies of protein refolding, how to renature mis-folded proteins avoiding aggregation is a usually encountered question.…”

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

“…Hence, some researchers paid close attention to on-column refolding techniques based on chromatography (Hamaker et al 1996). These techniques included affinity chromatography (e.g.…”