Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

Agricultural Service Center Palatinate (DLR Rheinpfalz), Department of Phytopathology, Laboratory for Biotechnological Crop Protection, Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany

Corresponding author: johannes.jehle@dlr.rlp.de

Abstract

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 °C, –20 °C, and –80 °C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 °C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at –20 °C and –80 °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 °C for four months showed a significantly decreasing activity measured as median lethal concentration (LC₅₀), whereas the protein activity declined less than 11-fold after two years storage at –20 °C. When stored at –80 °C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC₅₀ values of 7–10 ng Cry1Ab per cm² 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 °C is recommended.