Heterologous Expression of GPT1 and ALG6 in S. pombe

gpt1/alg6 S. pombe (Sp61G4A (h-, ade6-M210, ade1, leu1-32, ura4-D18, gpt1::ura4-D1684, alg6::ura4+) was used for heterologous expression (Fanchiotti et al. 1998 (link)). S. pombe cells were grown at 28° in YEA medium or MM medium supplemented with adenine or leucine as needed (Moreno et al. 1991 (link)). Escherichia coli strain STBL3 (Invitrogen, Carlsbad, CA) was grown in LB medium with 100 µg/ml ampicillin when needed. Reagents for yeast media were obtained from Difco Laboratories (Detroit, MI). N-Methyl-1-deoxynojirimycin (NMDNJ) was from Research Chemicals (North York, ON, Canada). Enzymes used for DNA procedures were from New England Biolabs (Ipswich, MA), KOD Hot Start DNA Polymerase was from Merck (Darmstadt, Alemania) andpCR2.1-TOPO Vector was from Invitrogen (Carlsbad, CA). Unless otherwise stated, all other reagents were from Sigma (St. Louis, MO). UDP-[¹⁴C]Glc was synthesized as previously reported with slight modifications (Wright A 1965). Protein concentrations were determined by Bio-Rad Protein Assay as described by the manufacturer.