Purification and characterization of an auxin-binding protein from Arabidopsis thaliana expressed in baculovirus-infected insect cells.
The auxin-binding protein At-ERabp1 is of very low abundance in Arabidopsis thaliana; it hinders any study at the protein level as it is difficult to collect large amounts from the plant. We therefore chose to express At-ERabp1 in baculovirus-infected insect cells. Recombinant baculoviruses were selected in yeast according to Patel et al. (Nucleic Acids Res. 20, 97-104, 1991). The recombinant protein was purified to homogeneity by a simple procedure involving an affinity step on a succinyl-concanavalin A column. Labeling with the photoactive auxin 5-N3-[7-3H]indole-3-acetic acid demonstrated that the baculovirus-expressed protein belongs to the auxin-binding protein family as deduced from its cDNA homology to a gene previously characterized in maize. The mature polypeptide migrates on SDS-PAGE with an apparent molecular mass of about 23 kDa, its NH2-leader sequence is properly processed, and it bears a high-mannose-type sugar moiety. All results are in agreement with information derived from the cDNA analysis. The possible role of a functional dimerization is also discussed.
Massotte
D
D
Max-Planck-Institut für Züchtungsforschung, Köln, Germany.
Fleig
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U
Palme
K
K
D000345:Affinity Labels; D000818:Animals; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D007210:Indoleacetic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D000345:Affinity Labels; D007210:Indoleacetic Acids; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C083846:auxin-binding protein 1