Figure 2.

Heterologous expression, purification, and enzymatic kinetics of the two GS isoforms identified, GiGS1 and GiGS2. A, Western blot of the expressed GiGS1 and GiGS2 in yeast. Whole cell protein extracts from the yeast transformed with GiGS1 and GiGS2 were fractionated on a denaturing 12% polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and detected with Anti-His(C-term)-HRP antibody. The yeast knockout mutants were independently transformed with the empty pYES2.1 vector or the vector fused with GiGS1 or GiGS2. Lanes a and c, The expression of GiGS on the medium without Gal; lanes b and d, the expression of GiGS on the medium with Gal; lane e, the expression of the empty vector on the medium with Gal (as a negative control). B, The purification stages of GiGS1 and GiGS2. Purified proteins were subjected to SDS-PAGE (12% NuPAGE Novex Bis-Tris Gel) and silver stained. Low-M_r markers are in lane f. Crude yeast extracts before purification were loaded in lane e, and successive elution fractions were loaded in lanes a to d. Each lane contained 1 μg of protein. C, Enzymatic kinetics for GiGS1 and GiGS2 detected by synthetase assay using the purified proteins. Biosynthetic GS activity assays were carried out by varying the concentration of Glu in the reaction mixture. Plots of 1/V versus [S] are shown. Velocity data, expressed as proportions of V_max, were based on the three biological replicates; absolute V_max values were 3.68 and 4.01 μm min⁻¹ mg⁻¹ for GiGS1 and GiGS2, respectively. Lineweaver-Burk plots of the data were obtained with GiGS1 (K_m = 1.87 mm, r² = 0.99) and GiGS2 (K_m = 3.80 mm, r² = 0.999).