REFOLDING OF LYSOZYME ASSISTED BY MOLECULAR CHAPERONES IMMOBILIZED IN CELLULOSE: THE OPERATIONAL CONDITIONS THAT AFFECT REFOLDING YIELDS
Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IBs). To recover the protein activity, the IBs are isolated, solubilized and refolded. The protein refolding processes play a major role in the production of recombinant proteins; thus, various methodologies have been implemented, including dilution, dialysis and column chromatography with or without the assistance of molecular chaperones. Recently, it was demonstrated that the apical domain of GroEL (AD), DsbA and DsbC immobilized on cellulose improved the efficiency of chromatographic refolding of rhodanese and lysozyme. Although immobilized chaperones and foldases greatly improve refolding yields, their use has been limited. To improve their potential use at the bioprocess scale, it is essential to understand the effects of operational conditions and additives on refolding yields. Therefore, we investigated the lysozyme refolding kinetics assisted by the apical domain of GroEL (AD), DsbA and DsbC in either soluble or immobilized on cellulose with different lysozyme concentrations, different chaperone:lysozyme ratios, absence of redox pairing, presence of glycerol and presence of high concentrations of GdnHCl and βmercaptoethanol (β-ME). Our results provide insight to improve the use of molecular chaperones in the refolding of recombinant proteins expressed as inclusion bodies.
Antonio-Pérez A., Rivera-Hernández T., Aldaz-Martínez M.L., Ortega-López J. (2012). Oxidative refolding of lysozyme assisted by the GroEL Apical Domain, DsbA and DsbC immobilized in cellulose. Biotechnology and Bioprocess Engineering 17, 703-710.
Antonio-Pérez A., Ramón-Luing L. A., Ortega-López J. (2012). Chromatographic refolding of rhodanese and lysozyme assisted by the GroEL apical domain of GroEL, DsbA, and DsbC immobilized in cellulose. Journal of Chromatography A, 1248, 122-129.
Baneyx F., Mujacic M. (2004). Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology 22, 1399-1408.
Basu A., Li X., Leong S. S. J. (2011). Refolding of proteins from inclusion bodies: rational design and recipes. Applied Microbiology and Biotechnology 92, 241-251.
Chow M.K. (2006). The REFOLD database: a tool for the optimization of protein expression and refolding. Nucleic Acids Research 34, D207- D212.
De Bernardez C. (1998). Refolding of recombinant proteins. Current Opinions in Structural Biology 9, 157-163.
Dong X.Y., Yang H., Sun Y. (2000). Lysozyme refolding with immobilized GroEL column chromatography. Journal of Chromatography A 878, 197-204.
Gekko K., Timasheff S.N. (1981). Thermodynamic and kinetic examination of protein stabilization by glycerol. Biochemistry 20, 4677-4686
Gupta P., Hall C. K., Voegler A. C. (1998). Effect of denaturant and protein concentrations upon protein refolding and aggregation: A simple lattice model. Protein Science 7, 2642-2652.
Hartl F.U., Hayer-Hartl M. (2009). Converging concepts of protein folding In vitro and In vivo. Nature Structural & Molecular Biology 16, 574- 581.
Jhamb K., Jawed A., Sahoo D.K. (2008). Immobilized chaperones: A productive alternative to refolding of bacterial inclusion body proteins. Process Biochemistry 43, 587- 597.
Jungbauer A., Kaar W. (2007). Current status of technical protein refolding. Journal of Biotechnology 128, 587-596.
Jungbauer A., Kaar W., Schlegl R. (2004). Folding and refolding of proteins in chromatographic beds. Current Opinions in Biotechnology 15, 487-596.
Koths K. (1995). Recombinant proteins for medical use: the attractions and challenges. Current Opinions in Biotechnology 6, 681-686.
Langenhof M., Leong S.S.J., Pattenden L. K., Middelberg A.P.J. (2005). Controlled oxidative protein refolding using an ion-exchange column. Journal of Chromatography A. 1069, 195-201.
Lu D., Liu Z. J. (2008). Dynamic redox environmentintensified disulfide bond shuffling for protein refolding In vitro: Molecular simulation and experimental validation. Physical Chemistry B 112, 15127-15133.
Lyles M. M., Gilbert H. F. (1991). Catalysis of the oxidative folding of ribonuclease by a protein disulfide isomerase: Dependence of the rate on the composition of the redox buffer. Biochemistry 30, 613-619.
Maskos K., Huber-Wunderlich M., Glockshuber R. (2003). DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns In vitro and In vivo. Journal of Molecular Biology 325, 495-513.
Middelberg A.R. (2002). Preparative protein refolding. Trends in Biotechnology 20, 437- 443.
Misawa S., Kumagai I. (1999). Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies. Peptide Science 51, 297-307.
Mizobata T., Kawagoe M., Hongo K., Nagai J., Kawata Y. (2000). Refolding of target proteins from a “rigid” mutant chaperonin demonstrates a minimal mechanism of chaperonin binding and release. Journal of Biological Chemistry 275, 25600-25607.
Ramon-Luing L.A., Cruz-Migoni A., Ruiz-Medrano R., Xoconostle-Cazares B., Ortega-Lopez J. (2006). One-step purification and immobilization in cellulose of the GroEL apical domain fused to a carbohydrate-binding module and its use in protein refolding. Biotechnology Letters 28, 301-307.
Ruoppolo M., Freedman R. B., Pucci P., Marino G. (1996). Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate. Biochemistry 34, 9380-9388.
Teshima T., Kohda J., Kondo A., Taguchi H., Yohda M., Fukuda H. (2000). Preparation of Thermus thermophiles holo-chaperoninimmobilized microspheres with high ability to facilitate protein refolding. Biotechnology and Bioengineering 68, 184-190.
Tsumoto K., Umetsu M., Yamada H., Ito T., Misawa S., Kumagai I. (2003). Practical considerations in refolding proteins from inclusion bodies. Protein Expression and Purification 28, 1-8.
Zapun A., Missiakas D., Raina S., Creighton T.E. (1995). Structural and functional characterization of dsbc, a protein involved in disulfide bond formation in Escherichia coli. Biochemistry 34, 5075-5089.
By publishing your paper in our journal you are also granting it the copyright of the information that it contains.