Purification and characterization of an auxin-binding protein from Arabidopsis thaliana expressed in baculovirus-infected insect cells.
Sleutelwoorden
Abstract
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.