Correlative e¡ecton the toxicityof three surface-exposed loops in the receptor-binding domain of theBacillus thuringiensis Cry4B


By jpeza - Posted on 21 Octubre 2009

Fecha Publicación: 
18 Sep 2009
Nombre de Revista: 
Datos del paper
Autor Principal: 
Tararat Khaokhiew
Volumen: 
300
Issue: 
1
Página Inicial: 
139
Página Final: 
145
Abstract: 

Surface-exposed loop residues, Pro389 (b6–b7 loop), Glu417 (b8–b9 loop), Tyr455
and Asn456 (b10–b11 loop), in the receptor-binding domain of the Bacillus
thuringiensis Cry4Ba toxin have been previously demonstrated to be crucial for
toxicity. Herein, five combinations of two-loop mutants, P389A/E417A (b6–b7/
b8–b9 loops), P389A/Y455A, P389A/N456A (b6–b7/b10–b11 loops), E417A/Y455A
and E417A/N456A (b8–b9/b10–b11 loops), were constructed as a means of
examining a correlative effect of these three critical loops on Cry4Ba toxicity. All
130-kDa mutant protoxins were overexpressed as inclusion bodies in Escherichia
coli with yields comparable to the wild-type toxin. In addition, all mutant toxins
were structurally stable upon solubilization and trypsin activation in carbonate
buffer, pH 9.0. Interestingly, E. coli cells expressing all the double-loop mutants
showed an almost complete loss in toxicity against Aedes aegypti mosquito larvae
while their corresponding single-loop mutants exhibited reduced activity of
approximately 50%. Moreover, in situ binding analysis revealed that the 65-kDa
purified toxins representing each pairwise mutant exhibited reduced binding to
apical microvilli of A. aegypti larval midgut when compared with the single
mutants. Altogether, the data demonstrate for the first time that all these three
surface-exposed loops of the Cry4Ba toxin are equally involved in receptor binding
and hence toxicity.

Dirección del Autor: 

Chamras Promptmas,
Department of Clinical Chemistry, Faculty of
Medical Technology, Mahidol University,
Bangkok-Noi, Bangkok 10700, Thailand.
Tel.: 1662 419 7168; fax: 1662 412 4110;
e-mail: mtcpm@mahidol.ac.th

Keywords: 
Bacillus thuringiensis; Double-loop mutants; Mosquito-larvicidal activity; In situ binding analysis.
Coautores: 

Chanan Angsuthanasombat ,  Chamras Promptmas