Cloning and expression of a 5-enolpyruvylshikimate-3-phosphate synthase gene from Halomonas variabilis.
A novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene of 1.35 kb was cloned from a cosmid library of Halomonas variabilis HTG7, inserted into vector pET-28a (+) and transformed in Escherichia coli BL21 (DE3). EPSPS was over-expressed in soluble form after induction with IPTG at 30 degrees C and it showed a single band in SDS-PAGE, which corresponds to a molecular weight of 51 kD. Deduced amino acid sequence analysis showed that there is little homology with the aroA genes which encode glyphosate-tolerant EPSPS in known sources, such as E. coli K12 and Agrobacterium sp. CP4. The over-expressed EPSPS was purified on nickel-nitrilotriacetic acid resin and detected by Western blotting analysis. Enzyme activity measurements demonstrated that there were 4.27 units/mg in cell extract, compared with 0.049 units/mg of the control. There is an 87-fold increase in specific activity for EPSPS.
Liu
Zhu
Z
Biotechnology Research Institute, Chinese Academy of Agriculture Science, Beijing 100081, People's Republic of China.
Pan
Zhiwei
Z
Xu
Yuquan
Y
Dong
Zhiyang
Z
Yang
Zhirong
Z
Lin
Min
M
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids / Q000378:metabolism; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D020632:Halomonas / Q000201:enzymology* / Q000235:genetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000097797:Glyphosate
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D004791:Enzyme Inhibitors; D005998:Glycine; D011993:Recombinant Fusion Proteins; D000097797:Glyphosate