Figure 3

The recombinant his-tagged wild-type and R38Q mutant 3-phosphoglycerate kinase was subjected to affinity purification on Ni-NTA column as described previously [20]. A. SDS-PAGE of recombinant wild-type and R38Q mutant S. cerevisiae 3-phosphoglycerate kinase. The proteins (1 and 1.8 μg respectively) was separated in 10% polyacrylamide gel and stained with Coommassie blue R250. B. TLC analysis of sugars prior to and after in-situ separation with R38Q. The recombinant R38Q mutant (R38Q-PGK) was coupled with Protein A sepharose beads and incubated overnight with a mixture of sugars, 3-phosphoglycerate (3PGA), ribulose-5-phosphate (R5P), Glucose-6-phosphate (G6P) and Fructose-6-posphate (F1,6-bP). After washing with 180 mM NaCl, the sugars were eluted with 1 M NaCl. Lane 1, mixture of sugar prior to incubation with R38Q-PGK and Lane-2 after elution with 1 M NaCl.