The screening of yeast expression and purification conditions for an UDP-GlcNac transporter

FAVAROLO BELEN; BREDESTON LM

Tucuman

Congreso; XLI Reunion Anual de la Sociedad Argentina de Biofisica; 2012

Sociedad Argentina de Biofisica

Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of Golgi apparatus, where glyconjugate biosynthesis take place by specific transferases. To these transport process an antiport mechanism, nucleotide-sugar/nucleoside monophosphate, have been proposed (Hirschberg at al, 1998). However the mechanism at the molecular level and the aminoacid residues involved in the binding and transport of substrates, are unknown. The main limitation to structure-function studies of NST is the production of high levels of recombinant protein. Recently (Drew et al 2007) was described a methodology, based on GFP fusion to membrane proteins, for a rapid screening of expression and purification conditions. In this work we studied the production of 10 NST from diverse origins (human, mouse, yeast, and worm) tagged to GFP in S. cerevisiae. Transformed cells were growth in media containing: 0.67% YNB, 0.08% Complete supplement mixture-URA, 0.1% glucose and induced with 2% galactose for 24hs at 28oC. Two of them, Hut1 from S. pombe (no characterized) and C03H5.2 from C. elegans (an UDP-GlcNac transporter) showed the higher expression levels measuring fluorescence from whole cells. Then we decided to study in details the optimum conditions of expression of C03H5.2 in 50 ml tubes containing 2 ml growth media. Results shown: i) differences in expression between transformed colonies; ii) not significantly changes in expression with the following conditions: addition of chemical chaperones (DMSO, glycerol, histidine), regulation of pH with phosphate buffer (pH 7.00), decrease the temperature to 20 oC, or increase the oxygen exchange with air pore tape sheets; iii) addition of 0.6% yeast extract and 0.1% peptone increase 4 times fluorescence yields. Preliminary attempts to purified C03H5.2-GFPhis8, by affinity chromatography from cells growth in 100 ml culture, showed a main band with a molecular mass corresponding to the fusion protein and a minor band corresponding to a N-terminal degradation fragment. 1. Hirschberg et al (1998) Ann. Rev. Biochemistry 2. Drew et al (2007) Nat. Protoc. 3, 784-98 Acknowledgment: supported by CONICET