Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana

Wilkie, S.E. and Warren, M.J. (1998) Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana. Protein Expr Purif, 12 (3). pp. 381-9. ISSN 1046-5928 . (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1006/prep.1997.0845

Abstract

Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana. cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods. Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species. Analysis of the recombinant proteins on denaturing PAGE gels indicated subunit Mrs of between 44 and 45 K. Kinetic parameters (Km and kcat) obtained for all four substrates (aspartate, alpha-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species. Further characterization of the purified recombinant enzymes alongside native enzymes from A. thaliana leaf tissue on AAT activity gels confirmed the identity of asp1 and asp2 as the mitochondrial and cytosolic AAT genes but indicated that asp5 may encode an amyloplastic rather than the chloroplastic enzyme.

Item Type: Article
Uncontrolled keywords: Amino Acid Sequence Arabidopsis/*enzymology/genetics Aspartate Aminotransferases/chemistry/*genetics/isolation & purification/metabolism Base Sequence DNA Primers/chemistry Electrophoresis, Polyacrylamide Gel Escherichia coli/enzymology/genetics Gene Expression Regulation, Enzymologic/*genetics Isoenzymes/chemistry/*genetics/isolation & purification/metabolism Kinetics Molecular Sequence Data Plant Proteins/chemistry/*genetics/isolation & purification/metabolism Polymerase Chain Reaction Recombinant Proteins/chemistry/genetics/isolation & purification/metabolism Sequence Alignment
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Martin Warren
Date Deposited: 15 Oct 2009 08:39
Last Modified: 14 Jan 2010 14:42
Resource URI: http://kar.kent.ac.uk/id/eprint/11079 (The current URI for this page, for reference purposes)
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