Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.
The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.
Pang
S Z
SZ
Department of Horticultural Sciences, Cornell University, Geneva 14456.
Oberhaus
S M
SM
Rasmussen
J L
JL
Knipple
D C
DC
Bloomquist
J R
JR
Dean
D H
DH
Bowman
K D
KD
Sanford
J C
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001681:Biological Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D012604:Scorpion Venoms; C068292:insectotoxin I5A