High-yield expression and purification of a monotopic membrane glycosyltransferase
2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, Vol. 66, no 2, 143-148 p.Article in journal (Refereed) Published
Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-Dodecyl-β-D-maltoside (DDM) (~1× critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.
Place, publisher, year, edition, pages
Elsevier , 2009. Vol. 66, no 2, 143-148 p.
Membrane protein, Monotopic, Overexpression, Optimization, Size-exclusion chromatography, Glycosyltransferase, Escherichia coli
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-29063DOI: 10.1016/j.pep.2009.03.008ISI: 000266394800004OAI: oai:DiVA.org:su-29063DiVA: diva2:229026