The expression and purification of the N-terminal activation domain of the transcription factor c-Myc: a model substrate for exploring ERK2 docking interactions.
ERK2 is a mitogen-activated protein kinase (MAPK) that plays pivotal roles in cell signal transduction, where it mediates effects on proliferation and differentiation by growth factors and hormones. An important substrate of ERK2 is the transcription factor c-Myc, which mediates cell cycle progression. The phosphorylation of Ser-62 on c-Myc by ERK2 is thought to contribute to the increased stability of c-Myc during the cell cycle and is thus a critical cellular event. However, the mode of c-Myc recognition by ERK2 is not understood. Early studies by Gupta and Davis concluded that ERK2 specificity determinants are located in residues 1-100 of c-Myc, its activation domain. To pursue both structural and kinetic studies a rapid, but efficient purification method, for the production of the activation domain of c-Myc from an Escherichia coli source, was developed. We chose the minimal number of high-resolution steps to maximize both yield and efficiency without sacrificing purity. Thus, GST-(c-MycDelta2-99)-His(6) was expressed in E. coli, and purified using glutathione-agarose affinity chromatography. Cleavage of the GST fusion protein by thrombin and subsequent purification by nickel-agarose affinity chromatography yielded 8 mg of purified (c-MycDelta2-99)-His(6) from one liter of LB culture. Rigorous characterization demonstrated that under standard assay conditions (c-MycDelta2-99)-His(6) is phosphorylated by ERK2 with the following Michaelis parameters: k(cat)=10.4s(-1), K(M)(c-Myc)=57.4 microM. In summary, a rapid procedure is outlined for the preparation of (c-MycDelta2-99)-His(6) that will be useful for mechanistic and biophysical studies of ERK2.
Waas
William F
WF
Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, TX 78712, USA.
Dalby
Kevin N
KN
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D012694:Serine; D006639:Histidine; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D013917:Thrombin