Purification of active chloroplast sedoheptulose-1,7-bisphosphatase expressed in Escherichia coli.
Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere. This makes SBPase an appropriate target for intensive study. We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag. The identity of the recombinant SBPases was confirmed by SDS-PAGE analysis and immunological detection with a specific antibody. Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography. Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity. Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts. The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography.
John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom. roy.dunford@bbsrc.ac.uk
R P
RP
Dunford
M A
MA
Catley
C A
CA
Raines
J C
JC
Lloyd
T A
TA
Dyer
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013403:Sugar Phosphates; D014908:Triticum / Q000201:enzymology / Q000235:genetics
D000906:Antibodies; D018744:DNA, Plant; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C022079:sedoheptulose 1,7-bisphosphate; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase