Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens.
Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.
Gao
Yong
Y
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA. ygao2@dow.com
Schafer
Barry W
BW
Collins
Randy A
RA
Herman
Rod A
RA
Xu
Xiaoping
X
Gilbert
Jeffrey R
JR
Ni
Weiting
W
Langer
Vickie L
VL
Tagliani
Laura A
LA
D000595:Amino Acid Sequence; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics*; D015870:Gene Expression; D006031:Glycosylation; D006460:Hemolysin Proteins; D007306:Insecticides / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics*; D011551:Pseudomonas fluorescens / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014357:Trypsin / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry / Q000235:genetics*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin