Protein Refolding Mediated by Reverse Micelles of Cibacron Blue F-3GA Modified Nonionic Surfactant

Abstract

An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery.

Versatility of Reverse Micelles: From Biomimetic Models to Nano (Bio)Sensor Design

“…In the past three decades, RMs have been used in different scientific fields for enzymatic catalysis [170], as models of membrane systems for the separation of proteins [171], for enzyme immobilization and storage of their activity [172,173], for studying changes in the protein structure and modeling intracellular crowding [174][175][176][177][178], for encapsulation of drugs and essential oils into hydrophilic water cores [179][180][181], for enhancing and tailoring the luminescent properties of fluorescent compounds [182][183][184][185], for protein refolding [186,187], and as nanoreactors [188][189][190] (Figure 9). Below, the potential of reverse micelles is highlighted for the developments of nanotechnology applied in fields of great interest, in addition to their contribution to opportunities in other domains.…”

An Approach for Increasing the Mass Recovery of Proteins Derived from Inclusion Bodies in Biotechnology

“…In this field many methods have demonstrated increases in bioactive recovery of proteins (Middelberg, 2002;Tsumoto et al, 2004;Wu et al, 2006;Prinsloo et al, 2006); however, a satisfactory result has not yet been obtained. Protein refolding by hydrophobic interaction chromatography (HIC) was intially reported in 1992 and is recognized as a good tool for protein folding (Geng and Chang, 1992).…”

Water Encapsulation by Nanomicelles

“…A fine-tuning of the experimental conditions allowed formation of a propofol trimer and tetramer with a water molecule and to determine the structure of the aggregates. Because of their important biological role, several applications have been found for RMs: they can be used as reaction systems for enzymatic catalysis, [5,6] drug delivery, [7,8] solvent-based extraction of proteins, [9,10] industrial processes, [11] microenvironment for discovery of protein structures, [12] protein refolding, [13,14] or even treated as models of membrane systems for the separation of proteins, [15] etc. Interpretation of the spectra in the light of high-level calculations allowed determination of the clusters structure and demonstration that the trimer of propofol with a water molecule forms cyclic hydrogen-bond networks but, on the other hand, the tetramer is big enough to encapsulate the water molecule inside its hydrophilic core.…”

“…[1,2] RMs are thus able to encapsulate microscopic pools of water while the apolar part remains in contact with an organic solvent. Because of their important biological role, several applications have been found for RMs: they can be used as reaction systems for enzymatic catalysis, [5,6] drug delivery, [7,8] solvent-based extraction of proteins, [9,10] industrial processes, [11] microenvironment for discovery of protein structures, [12] protein refolding, [13,14] or even treated as models of membrane systems for the separation of proteins, [15] etc. Because of their important biological role, several applications have been found for RMs: they can be used as reaction systems for enzymatic catalysis, [5,6] drug delivery, [7,8] solvent-based extraction of proteins, [9,10] industrial processes, [11] microenvironment for discovery of protein structures, [12] protein refolding, [13,14] or even treated as models of membrane systems for the separation of proteins, [15] etc.…”