Protein refolding at high pressure: Optimization using eGFP as a model

• Published in: Process biochemistry (1991) Vol. 46; no. 2; pp. 512 - 518
• Main Authors: Malavasi, N.V., Foguel, D., Bonafe, C.F.S., Braga, C.A.C.A., Chura-Chambi, R.M., Vieira, J.M., Morganti, L.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01-02-2011
• Subjects: Aggregation; atmospheric pressure; bioactive properties; dissociation; fluorescence; Green fluorescent protein; High hydrostatic pressure; high pressure treatment; inclusion bodies; Inclusion body; monitoring; mutants; recombinant proteins; Refolding; salts; solubility; yields; Aggregation; High hydrostatic pressure; Inclusion body; Refolding; Green fluorescent protein
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2010.10.002

Refolding of a mutant form of green fluorescent protein (eGFP), which only emits characteristic fluorescence when in the natively folded state, was accomplished under high hydrostatic pressure (HHP). Compression of eGFP inclusion bodies (IB) at 2.40kbar for 30min dissociated most of the aggregates and reduced the quantity of IBs. However, fluorescence at 509nm indicated that eGFP did not refold under this condition. The refolding process was evaluated under various decompression conditions, following IB dissociation at 2.40kbar. During stepwise decompression, increases in fluorescence were obtained at pressures ranging between 1.38kbar and atmospheric pressure. The highest levels of eGFP refolding were achieved by incubation at pressure levels between 0.35 and 0.69kbar in the absence of chaotropic reagents. The refolding was abolished when HHP was applied in the presence of 0.5–1.5M GdnHCl. Our approach focused on monitoring the bioactivity of the recombinant protein, i.e., fluorescence, instead of solubility, which is not an ideal indicator of proper refolding. The higher yields of a bioactive product by incubation at pressure levels of 0.35–0.69kbar without using chaotropic salts improve upon the HHP-refolding methods that have been previously described.