Purification of Membrane Proteins Using a Micropreparative Gel Electrophoresis Apparatus: Purification of Subunits of the Integral Membrane Protein Bacillus subtilis aa3-Type Quinol Oxidase for Low Level Amino Acid Sequence Analysis

Abstract

We have developed a continuous elution micropreparative gel electrophoresis system in which small amounts of proteins, e.g., 1-10 μg, are continuously eluted from one-dimensional polyacrylamide gel columns in either native or denatured form. Depending on the electrophoretical parameters proteins are separated according to their size and/or net charge. The system is based on a simple modification of the Mini-Protean IT 2D unit from Bio-Rad Inc. The apparatus is connected on-line to a high-performance liquid chromatograph allowing accurate delivery of the elution solvent and direct analysis of the separated samples by uv detection. The high resolution of the apparatus allows the purification of individual proteins, even from complex mixtures, in only one step, with overall recoveries of approximately 70%. In addition to standard proteins the system has been applied to the purification of the membrane-bound and thus highly hydrophobic subunits I and II from Bacillus subtilis aa3-type quinol oxidase. The hydrophobic character of these subunits has hindered their purification by other conventional methods. Isolated subunits were collected directly into a buffer suitable for proteolytic digestion and automated amino acid sequence analysis. The internal amino acid sequences determined could all be found in the DNA sequence recently reported by Santana et al. (J. Biol. Chem. 267, 10225-10231, 1992), thereby confirming the expression of such an oxidase.