PURIFICATION AND CHARACTERIZATION OF AN AUXIN-BINDING PROTEIN FROM ARABIDOPSIS-THALIANA EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS

被引：11

作者：

MASSOTTE, D ^{[1
]}

FLEIG, U ^{[1
]}

PALME, K ^{[1
]}

机构：

[1] MAX PLANCK INST ZUCHTUNGSFORSCH,D-50829 COLOGNE,GERMANY

来源：

PROTEIN EXPRESSION AND PURIFICATION | 1995年 / 6卷 / 03期

关键词：

D O I：

10.1006/prep.1995.1028

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The auxin-binding protein At-ERabpl 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-ERabpl 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-N-3-[7-H-3]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. (C) 1995 Academic Press, Inc.

引用

页码：220 / 227

页数：8

共 30 条

[1]

Hesse Feldwisch T.J., Balshilsemann D., Bauw G., Puype M., Vandekerckhove J., Lobler M., Klambt D., Schell J., Palme K., Molecular cloning and structural analysis of a gene fromzea mays (L.) coding for a putative receptor for the plant hormone auxin, EMBO J, 8, pp. 2453-2461, (1989)

[2]

Lobler M., Klambt D., Auxin-binding proteins of corn (Zea mays L.) I. Purification by immunological methods and characterization, J. Biol. Chem, 260, pp. 9848-9853, (1985)

[3]

Campos N., Feldwisch J., Zettl R., Boland W., Schell J., Palme K., Identification of auxin-binding proteins using an improved assay for photoaffinity labeling with 5-lV

[4]

r(7-: H)-indole-3-acetic acid, Technique, 3, pp. 69-75, (1991)

[5]

Venis M.A., Napier R.M., Barbier-Brygoo H., Maurel C., Perot-Rechmann C., Guern J., Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity, Proc. Natl. Acad. Sci. USA, 89, pp. 7208-7212, (1992)

[6]

Palme K., Hesse T., Campos N., Garbers C., Yanofsky M.F., Schell J., Molecular analysis of an auxin binding protein gene located on chromosome 4 of Arabidopsis, Plant Cell, 4, pp. 193-201, (1992)

[7]

Schertler G.F.X., Overproduction of membrane proteins, Curr. Opin. Struct. Biol, 2, pp. 534-544, (1992)

[8]

Miller L.K., Baculoviruses: High-level expression in insect cells, Curr. Opin. Gen. Dev, 3, pp. 97-101, (1993)

[9]

O'reilly D.R., Miller L.K., Luckow V.A., Baculovi-rus Expression Vectors, (1992)

[10]

Davidson D.J., Castellino F.J., Asparagine-linked oligosaccharide processing in lepidopteran insect cells: Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells, Biochemistry, 30, pp. 6168-6174, (1991)

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