ISOLATED DOMAIN-II AND DOMAIN-III FROM THE BACILLUS-THURINGIENSIS CRY1AB DELTA-ENDOTOXIN BINDS TO LEPIDOPTERAN MIDGUT MEMBRANES

The DNA fragment encoding Cry1Ab domain II-III (45.3 kDa) was cloned a nd expressed, Domain II-III is expressed in low yields, In vitro bindi ng analysis to Manduca sexta and Trichoplusia ni larval midgut tissue sections demonstrated that domain II-III fragment bound along the micr ovilli of the midgut epithelium, indicating that this fragment retains binding functionality in the absence of domain I, Binding of domain I I-III to the midgut brush border membrane proteins from T. ni larvae i ndicated that Cry1Ab toxin and domain II-III bind to the same 150 kDa protein, In contrast, in M. sexta membranes, Cry1Ab toxin binds to 200 and 120 kDa proteins, and domain II-III only binds to the 200 kDa pro tein, Finally, binding assays with isolated brush border membrane vesi cles showed that the interaction of domain II-III with the membrane ve sicles is highly reversible, supporting the proposition that the integ ration of domain I into the membrane could participate in the irrevers ible binding of the toxin, These studies confirm that this part of the toxin is involved in binding interactions and could be separated as a discrete fragment that conserves at least part of its functionality. (C) 1997 Federation of European Biochemical Societies.