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Abstract:

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO2 and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl-β-d-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg2+ but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg2+ (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg2+ as cofactor and the inhibition by Ca2+. Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves. © 1995 Kluwer Academic Publishers.

Registro:

Documento: Artículo
Título:High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves
Autor:Rodriguez-Suarez, R.J.; Wolosiuk, R.A.
Filiación:Instituto de Investigaciones Bioquímicas (Fundación Campomar, IIBBA-CONICET, FCEN-UBA), Antonio Machado 151, Buenos Aires, 1405, Argentina
Palabras clave:Brassica napus; chloroplast; fructose-1,6-bisphosphatase; rapeseed; recombinant enzyme; reductive activation
Año:1995
Volumen:46
Número:1-2
Página de inicio:313
Página de fin:322
DOI: http://dx.doi.org/10.1007/BF00020446
Título revista:Photosynthesis Research
Título revista abreviado:Photosynth Res
ISSN:01668595
CODEN:PHRSD
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01668595_v46_n1-2_p313_RodriguezSuarez

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Citas:

---------- APA ----------
Rodriguez-Suarez, R.J. & Wolosiuk, R.A. (1995) . High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves. Photosynthesis Research, 46(1-2), 313-322.
http://dx.doi.org/10.1007/BF00020446
---------- CHICAGO ----------
Rodriguez-Suarez, R.J., Wolosiuk, R.A. "High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves" . Photosynthesis Research 46, no. 1-2 (1995) : 313-322.
http://dx.doi.org/10.1007/BF00020446
---------- MLA ----------
Rodriguez-Suarez, R.J., Wolosiuk, R.A. "High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves" . Photosynthesis Research, vol. 46, no. 1-2, 1995, pp. 313-322.
http://dx.doi.org/10.1007/BF00020446
---------- VANCOUVER ----------
Rodriguez-Suarez, R.J., Wolosiuk, R.A. High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves. Photosynth Res. 1995;46(1-2):313-322.
http://dx.doi.org/10.1007/BF00020446