Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2.
Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.
Hui
Catherine M
CM
Department of Biochemistry, University of Alberta, 561 Medical Sciences Building, Edmonton, Alta., Canada T6G 2H7.
Campistrous
Ana
A
Stuart
David T
DT
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D006160:Guanosine Triphosphate / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008540:Meiosis; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018411:Spodoptera
D018797:Cell Cycle Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D011494:Protein Kinases; C064979:IME2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases