Solubilization and Reconstitution of Membrane Proteins of Escherichia coli Using Alkanoyl-N-Methylglucamides1

Abstract

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

Telomerase Activity Reconstituted in Vitro with Purified Human Telomerase Reverse Transcriptase and Human Telomerase RNA Component

“…However, we found that FLAG-hTERT could be solubilized in buffer containing a high salt concentration and the nonionic detergent MEGA-9 (29,30). We exploited this property to partially purify the FLAG-hTERT proteins first using buffers in which most cellular protein could be solubilized ( Fig.…”

“…The recombinant FLAGhTERT protein was recovered at 2.0 mg/10 8 infected cells, a level not much different from those of other recombinant proteins. hTERT could be solubilized only with the nonionic detergent MEGA-9 in the presence of high salt concentrations (29,30).…”

Preparation and Properties of Immune-stimulating Complexes Containing Hepatitis B Virus Surface Antigen

“…Alternatively the following preparation method was used: 1 mg of HBsAg in 0.5 ml TN buffer was solubilized in 1 ~ of the dialysable detergent N-decanoyl-N-methyl-glycoside (Mega-10) (Hildreth, 1982;Hanatani et al, 1984) for 18 h at 37 °C. After incubation the mixture was supplemented with 0.05 ~ Quil A, 0.25 mg phosphatidylcholine and 0.25 mg cholesterol.…”

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

“…For example up to 2 mM (or more) stock solutions of batyl or chimyl alcohols in 0.8% (23 mM) of Mega-10 in water or 0.1 M tris-HCl buffer (pH 7.5) at 25ЊC were solubilized by sonication, and the solutions were clear for several hours at 25ЊC. 192 The CMC (critical micelle concentration) 193 of Mega-10 was found to be 7.44 mM and 7.15 mM in water (pH 7.4) and in 0.1 M Tris-HCl buffer (pH 7.5), respectively, 194,195 and decreased to 5.72 mM (0.1 M Tris-HCl, pH 7.5) when 0.1 mM batyl alcohol was present in solution. With the ionic detergents 21 and 22, 0.1 mM batyl alcohol decreased or increased the CMC values depending on the chain length n. 191 From the evidence presented it was concluded that glyceryl ethers were solubilized in aqueous medium by forming mixed micelles with the soluble detergents and that the solutions contained a mixture of micelles of the detergent alone and mixed micelles of detergent and glyceryl ether, and that the concentration of free glyceryl ether in solution was usually negligible.…”