Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays.
The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 degrees C, -20 degrees C, and -80 degrees C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 degrees C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at -20 degrees C and -80 degrees C was observed. Bioassays with neonate larvae of Ostrinia nubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 degrees C for four months showed a significantly decreasing activity measured as median lethal concentration (LC(50)), whereas the protein activity declined less than 11-fold after two years storage at -20 degrees C. When stored at -80 degrees C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC(50) values of 7-10 ng Cry1Ab per cm(2) diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at -80 degrees C is recommended.
Nguyen
Hang Thu
HT
Agricultural Service Center Palatinate (DLR Rheinpfalz), Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Neustadt an der Weinstrasse, Germany.
Jehle
Johannes A
JA
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D004355:Drug Stability; D004731:Endotoxins / Q000737:chemistry* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000494:pharmacology; D007313:Insecta / Q000187:drug effects*; D007306:Insecticides / Q000737:chemistry* / Q000494:pharmacology; D013048:Specimen Handling / Q000379:methods
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis