Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber

被引：24

作者：

Chen, XY ^{[1
]}

Reddanna, P ^{[1
]}

Reddy, GR ^{[1
]}

Kidd, R ^{[1
]}

Hildenbrandt, G ^{[1
]}

Reddy, CC ^{[1
]}

机构：

[1] Penn State Univ, Dept Vet Sci, Ctr Mol Toxicol, University Pk, PA 16802 USA

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 1998年 / 243卷 / 02期

关键词：

D O I：

10.1006/bbrc.1998.8115

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs. We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column. Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate. The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX. When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences. (C) 1998 Academic Press.

引用

页码：438 / 443

页数：6

共 20 条

[1] SEQUENCE OF A CDNA CLONE ENCODING A POTATO (SOLANUM-TUBEROSUM) TUBER LIPOXYGENASE [J].

CASEY, R .

PLANT PHYSIOLOGY, 1995, 107 (01) :265-266

[2] SPECTROSCOPIC DETERMINATION OF TRYPTOPHAN AND TYROSINE IN PROTEINS [J].

EDELHOCH, H .

BIOCHEMISTRY, 1967, 6 (07) :1948-&

[3] ELECTROPHORETIC ANALYSIS OF MAJOR POLYPEPTIDES OF HUMAN ERYTHROCYTE MEMBRANE [J].

FAIRBANKS, G ;

STECK, TL ;

WALLACH, DFH .

BIOCHEMISTRY, 1971, 10 (13) :2606-+

[4] 5-LIPOXYGENASE [J].

FORDHUTCHINSON, AW ;

GRESSER, M ;

YOUNG, RN .

ANNUAL REVIEW OF BIOCHEMISTRY, 1994, 63 :383-417

[5] EXPRESSION OF LIPOXYGENASE IN WOUNDED TUBERS OF SOLANUM-TUBEROSUM L [J].

GEERTS, A ;

FELTKAMP, D ;

ROSAHL, S .

PLANT PHYSIOLOGY, 1994, 105 (01) :269-277

[6] CALCULATION OF PROTEIN EXTINCTION COEFFICIENTS FROM AMINO-ACID SEQUENCE DATA [J].

GILL, SC ;

VONHIPPEL, PH .

ANALYTICAL BIOCHEMISTRY, 1989, 182 (02) :319-326

[7] CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].

LAEMMLI, UK .

NATURE, 1970, 227 (5259) :680-+

[8]

LECUYER TJ, 1993, BIOTECHNIQUES, V14, P436

[9]

MALIYAKAL JE, 1992, NUCLEIC ACIDS RES, V20, P2381

[10] Crystal structure of soybean lipoxygenase L-l at 1.4 angstrom resolution [J].

Minor, W ;

Steczko, J ;

Stec, B ;

Otwinowski, Z ;

Bolin, JT ;

Walter, R ;

Axelrod, B .

BIOCHEMISTRY, 1996, 35 (33) :10687-10701

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