Overexpression in Escherichia coli and Characterization of the Chloroplast Fructose-1,6-bisphosphatase from Wheat

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In this Subject: <a title="Anatomy & Physiology" href="/content/subcat?j_subject=157&j_availability=">Anatomy & Physiology
By this author: Tang G-L. ;&nbsp; Wang Y-F. ;&nbsp; Bao J-S. ;&nbsp; Chen H-B.

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)

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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 prime -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 M, and V_max 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.