engineered E. coli strains (Bartnicki et al. 1993a,b; Sammons 1994a; Lee et al. 1995).

  • To test for digestibility of the microbially-produced Bt proteins by simulated gastric and intestinal fluids (Bartnicki et al. 1993b; Keck et al. 1993; Ream 1994b,c).

  • To test for acute effects on mice of the microbially-produced Bt proteins (Naylor 1992, 1993a,b; Sammons 1994b).

Equivalency

Determining chemical or functional equivalence of two proteins is difficult, especially if there is any question about potential post-transcriptional modification of either protein. However, a battery of well-conceived biochemical and functional tests can provide reasonable assurance of equivalence. Tests performed on the plant and microbially-produced Bt proteins included:

  • Assessment of equal migration of the full length and trypsinated forms on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and binding of the migrated proteins to a polyclonal antibody.

  • Demonstration of lack of glycosylation.

  • Assessment of sequence similarity of about 10-15 amino terminal amino acids and up to three short internal protein sequences.

  • Determination of similar toxicities of the proteins to one or two insect species.

Protein reactivity with antibodies and protein mobility are ways to determine if two proteins are similar in structure. In SDS-PAGE immunoblotting tests of comigration, the plant- and microbially-produced Bt proteins always had similar, strong protein bands at the same gel position that reacted with the polyclonal antibody.2 However, in many cases, there were additional bands in the gel lanes with plant-expressed Bt protein or microbially-expressed Bt protein. These additional bands differed in migration, but they bound to the polyclonal antibody. That indicates that the antibody was not highly specific or that there were different Bt proteins in the two types of preparations. Inasmuch as there was less dissimilarity in the trypsinated preparations, some of the nonhomologous bands in the full-length preparations might have been biologically unimportant. In some comparisons for cotton, a non-Bt plant-protein extrac-

2  

The polyclonal antibody was raised against E. coli-produced recombinant Bt protein.



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