Production and Purification of a GFP-Tagged ABC Transporter CaCdr1p

• DOI: 10.1007/978-1-0716-2368-8_9
• Publication Date: 2022-06-30T16:21:33Z
• Authors: Jorgaq Pata; Alexis Moreno; Sandrine Magnard; Atanu Banerjee; Rajendra Prasad; Pierre Falson
• Keywords: 0301 basic medicine; 0303 health sciences; 03 medical and health sciences; Candida albicans; Green Fluorescent Proteins; Membrane Proteins; ATP-Binding Cassette Transporters; Saccharomyces cerevisiae

The production and purification are the first steps required in any functional or structural study of a protein of interest. In the case of membrane proteins, these tasks can be difficult due to low expression levels and the necessity to extract them from their membrane environment. This chapter describes a convenient method based on GFP tagged to the membrane protein to facilitates these steps. Production is carried out in the yeast S. cerevisiae and purification steps are carried out and monitored taking advantage of an anti-GFP nanobody. We show how GFP can be a very helpful tool for controlling the correct addressing of the protein and for probing and optimizing purification. These methods are described here for producing and purifying CaCdr1p, an ABC exporter conferring multiantifungal resistance to C. albicans. This purification method can be amenable to any other GFP-tagged protein.