A rapid and inexpensive method for the production of protein is crucial for the biopharmaceutical industry. The bottleneck in the case of genetically engineered systems lies in the recovery of active and properly refolded proteins from inclusion bodies. The correct refolding of a protein from its denatured state is dependent on several parameters including the concentration of the protein, the refolding buffers utilized, the approach employed and finally the type of refolding additives, if any, used in the process.
In this study, an emerging class of refolding additives, namely, ionic liquids were investigated for the refolding of denatured lysozyme protein. Lysozyme was used as a model system due to its rapid refolding kinetics and the fact that enzyme based assay could be carried out to measure the refolding yields. The ionic liquids selection was hypothesized based on the selection of cation and anion that constitute the ionic liquid. The efficacy of Imidazolium based ionic liquids as refolding additives was compared to conventional refolding approach that contains urea and redox-couple cysteine/cystine. Using response surface methodology (RSM) and central composite design (CCD), an empirical model was developed and validated by experimental results. Different refolding strategies were also evaluated; dilution refolding, adsorptive on column packed bed refolding and adsorptive on column fluidized bed refolding.
Experimental results indicate that in comparison to the conventional refolding buffer, among different ionic liquids examined, ionic liquid [EMIM]Cl has a pronounced effect on the refolding yield and time required to refold the denatured lysozyme. The optimal conditions were identified in this study as 150 μg/ml of denatured lysozyme in presence of 75 mM [EMIM] Cl in 50 mM HEPES buffer (pH 7.5). On-column refolding studies indicate that in comparison to the packed bed system, the fluidized bed ion-exchange system approach, with [EMIM] Cl as the elution-refolding buffer could tolerate higher concentration of denatured lysozyme (up to 25 mg) with refolding yield ca. 90% and fractional mass recovery ca. 82%. This was due to potentially lower mass transfer limitations in the fluidized bed compared to the packed bed. Thus, the versatility of ionic liquids coupled with on-column adsorptive refolding can possibly be the solution to the large scale downstream processing of proteins
