Overexpression in Escherichia coli and characterization of the chloroplast fructose-1,6-bisphosphatase from wheat.
An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Feng Lin Lu, Shanghai, 200032, People's Republic of China.
G L
GL
Tang
Y F
YF
Wang
J S
JS
Bao
H B
HB
Chen
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D006597:Fructose-Bisphosphatase