Useful tools for signal transduction research and drug screening
Quinn Lu Chao-Feng Zheng
Stratagene Cloning Systems, Inc.
Stratagene has successfully expressed and purified recombinant, wild-type human MEK1WT and a constitutively active mutant (MEK1CA) using the ESP yeast protein expression and purification system. These MEKs, produced in yeast, are full length and undegraded, whereas MEKs produced in E. coli are significantly degraded. Wild-type MEK1WT can be used as a substrate for upstream MEK activators, such as Raf, MEKK or mos. The uses for constitutively active MEK1CA include identifying potential substrates, making activated MAPK, screening for inhibitors and performing microinjection studies.
The term mitogen-activated protein kinase/extracellular signal-activated protein kinase (MAPK/ERK kinase, MEK) refers to two dual-specificity protein kinases that phosphorylate and thereby activate both threonine and tyrosine residues of mitogen-activated protein kinases (MAPK).1-3 In order to be fully active, MEK itself needs to be phosphorylated on two serine residues by other protein kinases, such as raf.4-6 The recombinant human MEKs produced in E. coli are not very active because E. coli lacks MEK activators. Substitution of the two phosphoacceptors (Ser-218 and Ser-222) in MEK1WT with glutamic acid, in combination with an internal deletion (from amino acid 32 to amino acid 51), produced a mutant MEK that is more active than wild type.4-6 This mutant, made by site-directed mutagenesis, is referred to as constitutively active MEK1 (MEK1CA) and does not need to be activated by other protein kinases.
Recombinant MEK1WT and MEK1CA are very useful tools for signal transductio n research and drug screening. Although MEK1WT can be expressed in soluble form in E. coli as glutathione-S-transferase (GST) fusions, these products are largely degraded.2, 7,8 Separating full-length protein from degradation products is difficult, presumably because of the dimeric nature of the glutathione-S-transferase moiety. In E. coli, the improper folding of proteins is one of the most common causes of inclusion body formation and degradation of recombinant proteins. One method for solving the problem of degradation is to express these proteins in eukaryotic cells, such as insect cells or yeast. In this article, we describe the expression of MEK1WT and MEK1CA as GST fusion proteins in Schizosaccharomyces pombe using the ESP expression and purification system.9 We show that full-length GST-MEK1WT and GST-MEK1CA can be produced in yeast and purified by GST affinity chromatography and that the recombinant MEKs produced in yeast retain full biological activity.
Stratagenes new ESP yeast protein expression and purification system (see page 72) provides a novel means for rapid, inexpensive and high-level production of proteins in S. pombe.9 In order to express MEK1WT and MEK1CA in S. pombe, the cDNAs coding for human MEK1WT and MEK1CA were subcloned into the pESP-1 expression vector at the BamH I site, in frame with GST. The expression constructs were transformed into the S. pombe host strain SP-Q01 (leu1-32h-). The cells were first grown in thiamine-containing YES medium and then in EMM medium, which does not contain thiamine, to induce the synthesis of GST-MEK1WT or GST-MEK1CA. For both GST-MEK1WT< /sup> and GST-MEK1CA, the expression level is about 8 mg per liter of induced culture.
GST-MEK1CA (figure 1) and GST-MEK1WT expressed in S. pombe were purified from crude cell extracts by a single GST affinity chromatography step, yielding about 4 mgs of purified protein per liter. The enzymes expressed and purified from S. pombe contained only full-length GST-MEK1CA, which is about 66 kDa (figure 1, panel B). In contrast, the preparation of GST-MEK1CA from E. coli contained numerous degradation products (figure 1, panel A), which were very difficult to remove.2
MEK1WT is a protein kinase that relays extracellular signals to mitogen-activated protein kinase (MAP kinase or ERK). By phosphorylating Thr-183 and Tyr-185, MEK1WT activates MAP kinase. To assure that the GST-MEK1WT and GST-MEK1CA expressed in S. pombe were active, we assessed their abilities to phosphorylate a recombinant MAP kinase and stimulate MAP kinase activity. As shown in figure 2, when GST-MEK1WT (lane 3) or MEK1CA (lane 4) were incubated with Nonactivated MAP Kinase in the presence of [g-32P]ATP, MAP kinase (ERK2) was phosphorylated in both reactions. Phosphorylation of ERK2 by GST-MEK1WT (lane 3) could only be seen upon prolonged exposure; the weak si gnal is a result of the lower activity of GST-MEK1WT as compared to GST-MEK1CA (lane 4).
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-MEK1WT or GST-MEK1CA (figure 2, lanes 5, 6). The autophosphorylation bands of GST-MEK1WT and GST-MEK1CA can be observed using increased amounts of kinase in the assay (not shown). Although both are active, the activity of GST-MEK1CA is much higher than that of GST-MEK1WT (lanes 3, 4 and lanes 5, 6), consistent with previous reports.4-6
Human MEK1WT and a constitutively active mutant of MEK1WT (MEK1CA) 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.
For expression in Stratagenes ESP protein expression and purification system, the S. pombe host strain SP-QO1 was transformed with pESP-MEK1CA 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 methylsulfonyl fluoride (PMSF), 100 units aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin]. GST-MEK1CA was purified as described.2, 10
For expression in E. coli, the entire coding sequence of MEK1CA was inserted at the BamH I site of the pGEX-2T plasmid. Epicurian Coli XL1-Blue cells containing pGEX-MEK1CA were induced by IPTG (1 mM) for 4 hours, when OD600 reached 0.6. Cells were then collected and broken by sonication in PBS containing 1% Triton X-100 and protease inhibitors (1 mM PMSF, 100 units aprotinin, 1 M pepstain A, 100 M of leupeptin and 1 g/ml of chymostatin). GST-MEK1CA was purified as described.2, 10
Assays for protein kinase activity were performed in 40 l of 25 mM HEPES buffer, pH 7.4, containing 50 mM ATP, 10 mM magnesium acetate, 10 mM b-mercaptoethanol and 2 mCi of [g-32P]ATP. Proteins were added to each reaction as indicated in figure 2, and the reactions were incubated at 30C for 15 minutes and then mixed with 40 l of 2x SDS-PAGE sample buffer.
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