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Title: Effects of different Brush Border Membrane Vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Author
item CHEN, LZ - CHINESE ACAD AG SCIENCES
item LIANG, GM - CHINESE ACAD AG SCIENCES
item Rector, Brian
item ZHANG, J - CHINESE ACAD AG SCIENCES
item WU, KM - CHINESE ACAD AG SCIENCES
item GUO, YY - CHINESE ACAD AG SCIENCES

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2008
Publication Date: 7/1/2008
Citation: Chen, L., Liang, G., Rector, B.G., Zhang, J., Wu, K., Guo, Y. 2008. Effects of different Brush Border Membrane Vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera. Insect Science, Vol 15, pp.497-503.

Interpretive Summary: Brush border membrane vesicles are small fragments of intestinal (or other organ) membranes that are used in laboratory tests to observe the direct interaction of an experimental substance (such as a protein or chemical) with the intestinal membrane, in place of using the live, intact animal. BBMVs are commonly used to study the interaction of Bacillus thuringiensis (Bt) toxins with insect midguts. In this study, several protocols for isolating and preparing BBMVs from midguts of Old World cotton bollworm, Helicoverpa armigera, were compared. Aspects of these protocols were integrated to provide a master protocol that maximizes the yield of high-quality BBMVs used to study differences between midguts of Bt-resistant and -susceptible H. armigera.

Technical Abstract: The brush border membrane vesicles (BBMV) isolated from midgut cells of insect have been widely used for studying of the binding receptors and action mode of Cry proteins produced by Bacillus thuringiensis (Bt). There are several methods for isolating insect BBMV used in one-dimension electrophoresis (i.e. SDS-PAGE). In recent years two-dimension (2D) electrophoresis is used in Bt receptors research, sample preparation is very important in 2D. In order to find out a suitable method to separate the larval BBMV of Helicoverpa amigera for 2D electrophoresis, three typical methods for BBMV preparation (Abdul-Rauf and Ellar’s method; English and Readdy’ method; Wolfersberger’s method) are compared. The results showed that all of these three methods did not affect the kinds and number of receptor proteins, while the quantity of proteins obtained separately by these methods were different. Protein quantity is very important for the research when insect number is limited such as resistance insect in the field. So we integrated these three isolated methods into one, to insure high quantity of BBMV proteins, which were proved to be suitable for 2D analysis.