Phosphatase-immunodepleted cell-free protein synthesis system.
A cell-free protein synthesis system using wheat-germ extract was improved by a novel approach involving selective removal of endogenous phosphatase, which reduces both the duration and the rate of translation by hydrolyzing ATP and GTP, from the translational reaction. Immunodepletion of the phosphatases by the antibodies raised against the major one of the wheat-germ phosphatase isozymes removed 20-40% of ATP-hydrolysis activity from the wheat-germ extract, and thereby prolonged the reaction period of translation. Moreover, the condensation of the phosphate-immunodepleted extract by polyethylene glycol (PEG) precipitation and the addition of copper ions, which was known to inhibit phosphatase and nuclease activity, increased the protein synthesis more than two-fold compared with the reaction using control IgG-treated condensed extract.
Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Japan.
Y
Y
Kawarasaki
H
H
Nakano
T
T
Yamane
D000255:Adenosine Triphosphate / Q000378:metabolism; D000906:Antibodies / Q000378:metabolism; D002457:Cell Extracts / Q000502:physiology; D003300:Copper / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics; D014908:Triticum / Q000201:enzymology*
D000906:Antibodies; D002457:Cell Extracts; D010940:Plant Proteins; D011092:Polyethylene Glycols; D003300:Copper; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases