Molecular cloning, functional expression in Escherichia coli, and characterization of multiple mitogen-activated-protein kinases from tobacco.
Eur J Biochem, 1995/10/01;233(1):249-57.
Wilson C[1], Anglmayer R, Vicente O, Heberle-Bors E
Affiliations
PMID: 7588752
Abstract
A screening of four tobacco cDNA libraries by PCR, using degenerate oligonucleotides corresponding to motifs conserved in mitogen-activated-protein kinases from animals and yeasts, resulted in the isolation of five different PCR fragments that showed high sequence similarity to mitogen-activated-protein kinases from other organisms. Full-length cDNAs were obtained for two of these, ntf4 and ntf6, and we have previously reported the isolation of one of the other cDNAs, ntf3 [Wilson, C., Eller, N., Gartner, A., Vicente, O. & Heberle-Bors, E. (1993) Plant Mol. Biol. 23, 543-551]. The three cDNAs, ntf3, ntf4 and ntf6, as well as a mutated form of ntf3, were fused to the glutathione-S-transferase gene and expressed as fusion proteins in Escherichia coli. All three wild-type recombinant proteins, with or without the glutathione-S-transferase fragment, are capable of autophosphorylation and phosphorylate myelin basic protein, in a reaction that is more strongly supported by Mn2+ than by Mg2+, while the kinase-negative Ntf3 mutant did not show any activity. Western-blot analysis showed that the recombinant proteins autophosphorylate on tyrosine residues and are recognized by antibodies prepared against mammalian mitogen-activated-protein kinases.
MeSH terms
Amino Acid Sequence; Base Sequence; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cloning, Molecular; DNA, Complementary; DNA, Plant; Escherichia coli; Gene Expression; Gene Library; Genes, Plant; Genetic Vectors; Molecular Sequence Data; Plants, Toxic; Polymerase Chain Reaction; Recombinant Proteins; Sequence Homology, Amino Acid; Nicotiana
More resources
EndNote: Download