Screening and Scale-Up of GLUT Transporter Constructs Suitable for Biochemical and Structural Studies

Identifying membrane proteins that can be produced and isolated in homogenous form in detergent is a lengthy trial-and-error process that can be facilitated by fluorescence-based screening approaches. We describe (1) the strategy and protocol of cloning by homologous recombination, (2) whole-cell and in-gel fluorescence measurements to estimate GLUT-GFP fusion protein yields, (3) use of size-exclusion chromatography monitored by fluorescence (FSEC) for assessing the homogeneity of the GLUT-GFP fusion proteins, and (4) the protocol for large-scale production and purification of the Bos taurus GLUT5 construct that enabled its crystal structure determination.