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

Liu, Yang; Dong, Xiaoyan; Sun, Yan

doi:10.1016/j.jcis.2005.04.017

Cited by 19 publications

(44 citation statements)

References 36 publications

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“…W 0 is not affected by pH at the extensive pH range (Figure 4), but it decreases exponentially with increasing NaCl concentration ( Figure 5). The latter is similar with the reversed micelles of CB-modified Span 85 27 and AOT. 39 The result indicates that weak electrostatic interactions also play an important role in the self-organization of the reverse micelle in the present nonionic surfactant system.…”

Section: Water Contentsupporting

confidence: 75%

“…27 From the readings of the titrator, water content (W 0 , the molar ratio of water to total surfactant concentration in the organic phase) was calculated.…”

Section: Methodsmentioning

confidence: 99%

“…The N ag and hydrodynamic radius (R h ) of the reverse micelles were, respectively, determined by static light scattering (SLS) and dynamic light scattering (DLS) at 25 AE 0.1 C. 27 The SLS and DLS measurements were made on Dawn EOS, Wyatt QELS, and Optilab DSP Instrument (Wyatt Technology Corp.). Reverse micellar solution was inline filtered through the 0.22 lm hydrophobic Millipore filter to remove any dust particles and flowed into a 70 lL quartz cell at 1.0 mL/min.…”

Section: Characterization Of Reverse Micellesmentioning

confidence: 99%

“…The reverse micellar phase behaviors such as water content, micellar size, and aggregation number (N ag ) were extensively investigated following the methods described in literatures. 7,20,27 Then, selective metal-chelate extraction of histidine-rich protein by a copper-chelate reverse micellar solution was successfully performed, and the stripping conditions for the recovery of the solubilized protein were determined.…”

Section: Introductionmentioning

confidence: 99%

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A metal‐chelate affinity reverse micellar system for protein extraction

Dong¹,

Meng²,

Feng³

et al. 2009

Biotechnology Progress

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A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X-45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X-45 less than 0.6, thermodynamically stable reverse micelles of water content (W(0)) up to 30 are formed. Di(2-ethylhexyl) phosphoric acid (HDEHP; 1-2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine-rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper-chelate reverse micelles (Cu-RM) are characterized for their W(0), hydrodynamic radius (R(h)), and aggregation number (N(ag)). Similar with reverse micelles of bis-2-ethylhexyl sodium sulfosuccinate (AOT), R(h) of the Cu-RM is also linearly related to W(0). However, N(ag) is determined to be 30-90 at W(0) of 5-30, only quarter to half of the AOT reverse micelles. Then, selective metal-chelate extraction of histidine-rich protein (myoglobin) by the Cu-RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine-tagged proteins expressed as inclusion bodies.

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Section: Water Contentsupporting

confidence: 75%

“…27 From the readings of the titrator, water content (W 0 , the molar ratio of water to total surfactant concentration in the organic phase) was calculated.…”

Section: Methodsmentioning

confidence: 99%

Section: Characterization Of Reverse Micellesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A metal‐chelate affinity reverse micellar system for protein extraction

Dong¹,

Meng²,

Feng³

et al. 2009

Biotechnology Progress

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“…With the affinity micelles, however, almost all the EGFP was solubilized to the organic phase and the extraction reached equilibrium in about 6 min of mixing operation. The kinetic behavior is similar to myoglobin extraction by Cu(II)-chelate reverse micelles, 19 but much faster than lysozyme and BSA extractions by the dye-ligand (Cibacron blue F-3GA) affinity reversed micelles (Span 85 in n-hexane), 17,25 although the same mixing condition was adopted. It has been well recognized that liquid-film mass transfer is the main resistance for protein solubilization into reverse micelles.…”

Section: Forward Extraction Of Purified Egfpmentioning

confidence: 79%

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Dong

Feng

Sun

2010

Biotechnology Progress

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Di(2-ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X-45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R(2). The Ni(II)-chelate reverse micelles were characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R(2) was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni(2+) ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two-stage purification by the metal-chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification.

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