( gnal is a result of the lower activity ...)Production of Full-Length, Biologically Active MEK1WT and MEK1CA

The activity of MAP kinase can be assessed by its ability to phosphorylate PHAS-I, a substrate developed by Stratagene.⁸ The activity of MAP kinase was increased dramatically in the presence of either GST-MEK1^WT or GST-MEK1^CA (figure 2, lanes 5, 6). The autophosphorylation bands of GST-MEK1^WT and GST-MEK1^CA can be observed using increased amounts of kinase in the assay (not shown). Although both are active, the activity of GST-MEK1^CA is much higher than that of GST-MEK1^WT (lanes 3, 4 and lanes 5, 6), consistent with previous reports.^4-6

Conclusions

Human MEK1^WT and a constitutively active mutant of MEK1^WT (MEK1^CA) were successfully produced using Stratagenes ESP yeast protein expression and purification system. These recombinant MEKs were active as MAP kinase activators. The MEKs purified from yeast were full length, whereas the MEK preparation from E. coli was predominantly composed of degraded products.

Methods

For expression in Stratagenes ESP protein expression and purification system, the S. pombe host strain SP-QO1 was transformed with pESP-MEK1^CA and grown in YES media at 30C to mid-log phase (when OD₆₀₀ reached 0.5). In order to deplete thiamine in the media and induce protein expression, the cells were collected by centrifugation, washed twice with water, resuspended in EMM media and grown at 30C for 18 to 20 hours. The cells were then collected and broken by vortexing with glass beads in sorbitol buffer (0.3 M sorbitol; 0.1 M NaCl; 5 mM MgCl₂ and 10 mM Tris-HCl, pH 7.4) containing protease inhibitors [1 mM phenyl
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