High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves
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...
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1995
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paper:paper_01668595_v46_n1-2_p313_RodriguezSuarez2023-06-08T15:16:10Z High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro Brassica napus chloroplast fructose-1,6-bisphosphatase rapeseed recombinant enzyme reductive activation 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. Fil:Rodriguez-Suarez, R.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1995 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01668595_v46_n1-2_p313_RodriguezSuarez http://hdl.handle.net/20.500.12110/paper_01668595_v46_n1-2_p313_RodriguezSuarez |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Brassica napus chloroplast fructose-1,6-bisphosphatase rapeseed recombinant enzyme reductive activation |
| spellingShingle |
Brassica napus chloroplast fructose-1,6-bisphosphatase rapeseed recombinant enzyme reductive activation Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| topic_facet |
Brassica napus chloroplast fructose-1,6-bisphosphatase rapeseed recombinant enzyme reductive activation |
| description |
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. |
| author |
Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro |
| author_facet |
Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro |
| author_sort |
Rodriguez Suarez, Roberto J. |
| title |
High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| title_short |
High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| title_full |
High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| title_fullStr |
High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| title_full_unstemmed |
High level expression in Escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (Brassica napus) leaves |
| title_sort |
high level expression in escherichia coli, purification and properties of chloroplast fructose-1,6-bisphosphatase from rapeseed (brassica napus) leaves |
| publishDate |
1995 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01668595_v46_n1-2_p313_RodriguezSuarez http://hdl.handle.net/20.500.12110/paper_01668595_v46_n1-2_p313_RodriguezSuarez |
| work_keys_str_mv |
AT rodriguezsuarezrobertoj highlevelexpressioninescherichiacolipurificationandpropertiesofchloroplastfructose16bisphosphatasefromrapeseedbrassicanapusleaves AT wolosiukricardoalejandro highlevelexpressioninescherichiacolipurificationandpropertiesofchloroplastfructose16bisphosphatasefromrapeseedbrassicanapusleaves |
| _version_ |
1768544952869978112 |