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 |
| |
Affiliation: | 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. |
| |
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, 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). |
| |
Keywords: | fructose-1,6-bisphosphatase wheat chloroplast expression characterization activation |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|