Overexpression in Escherichia coli and Characterization of the Chloroplast Fructose-1,6-bisphosphatase from Wheat
Authors: Tang G-L.; Wang Y-F.; Bao J-S.; Chen H-B.
Source: Protein Expression and Purification, Volume 19, Number 3, August 2000 , pp. 411-418(8)
Publisher: Academic Press
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Abstract:
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, Kcat is 22.9/S, Km is 121 
M, and Vmax is 128 mol/min · mg. The recombinant FBPase can be activated by DTT, Na+, or low concentrations of Li+, Ca2+, Zn2+, GuHCl, and urea, while it can be inhibited by K+ or NH+4. Copyright 2000 Academic Press.
Keywords: fructose-1,6-bisphosphatase; wheat; chloroplast; expression; characterization; activation
Language: English
Document Type: Research article
Affiliations: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Feng Lin Lu, Shanghai, 200032, People's Republic of China:
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