A metal-chelate affinity reverse micellar system for protein extraction.

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.

Synthesis, biocompatibility and cell labeling of L-arginine-functional beta-cyclodextrin-modified quantum dot probes.

A series of quantum dots (QDs), CdSe, CdSe/CdS and CdSe/ZnSe, coated with L-arginine-modified beta-cyclodextrin (beta-CD-L-Arg) were prepared in a solution of H2O and hexane by ultrasonic method and characterized using PL, UV-vis, TEM, EDX and FTIR techniques. We observed that beta-CD-L-Arg-coated QDs are water-soluble and stable with high colloidal properties in water. Their photophysical properties are similar to those of trioctylphosphine oxide (TOPO)-coated nanocrystals. The quantum yield (QY) of beta-CD-L-Arg/CdSe/ZnSe QDs in water is 68%, which is much higher than those of beta-CD-L-Arg/CdSe/CdS (26%) and beta-CD-L-Arg/CdSe (13%). The in vitro cytotoxicity of these QDs was evaluated in ECV-304, SH-SY5Y and Hela cells and low cytotoxicity was observed. In particular, the beta-CD-L-Arg/CdSe/ZnSe QDs presented lower cytotoxicity to these cells (CC(50) value is 173 microg/mL in ECV-304 cells for 48h). This may be due to the presence of the ZnSe and beta-CD-L-Arg outlayer, which may improve the biocompatibility of QDs. The QDs were further investigated for biological labeling in ECV-304 cells using confocal laser scanning fluorescence microscopy. We found that these QDs were capable of localing to the cytoplasm of cells. These results demonstrate that the beta-CD-L-Arg-coated QDs could be used as a potential photoluminescent nanocrystal probing agent with good biocompatibility.