Protein Separation Using Affinity-Based Reversed Micelles

Abstract

Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

Effect of hexanol on the reversed micelles of Span 85 modified with Cibacron Blue F-3GA for protein solubilization

“…In general, the K o a b values were about two orders of magnitude less than the K o a f values. This is consistent with the results obtained with traditional ionic surfactant based reversed micellar extractions [12,21] as well as those with the affinitybased reversed micellar system composed of CB-modified lecithin [22]. The slow back extraction has been considered due to the interfacial resistance to release a protein at the oilwater interface.…”

“…The readings from the Karl Fisher titrator, given as percent weight of water in the sample, were then converted to the water content, W 0 (molar ratio of water to Span 85 in organic phase). The concentration of the coupled CB in organic phase or free CB in aqueous phase was measured by VIS spectrophotometer at 605 nm [22]. Aqueous phase protein concentration was determined spectrophotometrically at 280 nm.…”

“…Considering a batch extraction process with a well-mixed two-phase system, and assuming that the two-film theory of mass transfer applies to the liquid-liquid extraction process [12,21,22], the mass transfer between the aqueous and micellar phases can be expressed by…”

Characterization of reversed micelles of Cibacron Blue F-3GA modified Span 85 for protein solubilization

“…However, antibody ligand is expensive and its instability may limit its application. Thus, chemical ligands are desired for their availability and applicability in harsh environments [22,23].…”

Chemical synthesis and crystallization of the dipeptide AcPheIleNH2 in TTAB/heptane/octanol reversed micelles

“…The ability to selectively solubilize polar and apolar compounds and the large contact area provided by the interface are two of the most interesting features of reversed micelles. Reversed micelles have been used as "nanoreactors" in numerous applications, ranging from enzymatic [1,2] and chemical catalysis [3][4][5], protein separation and refolding [6,7], synthesis of nanoparticles and quantum dots [8][9][10], drug delivery [11,12] and analytical methodologies [13,14]. * The synthesis of a peptide (amide) bond involves the condensation of an amine group of one amino acid and a carboxyl group of another.…”