Orally active androctonus amoreuxi pesticidal biopeptides
a biopeptide, orally active technology, applied in the field of natural-occurring pesticides, can solve the problems of chemical agents that are constantly under scrutiny, can be toxic to insect pests, and various forms of bt toxins, so as to enhance the effectiveness of baculovirus, enhance plant pest resistance, and increase the efficiency of virus action
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example 1
Fractionation of Polypeptides From Arthropod Venom
[0159]Polypeptides were enriched from arthropod venom using a variety of HPLC chromatography conditions. Crude arthropod venom was applied to either HP1100 or Microbore LC columns as indicated. The peptides were resolved using a method set forth herein. The fractions were assayed for pesticidal activity as described elsewhere herein.
[0160]For all HPLC chromatography methods Solvent A consists of 95% Water and Solvent B consists of 95% acetonitrile with either 0.1% TFA or HFBA as noted.
[0161]For gradients over HP1100 columns a 0.6 mL / min flow rate was used unless otherwise noted.
METHOD 1:60% Solvent B in 70 minutesTFA; C4 columnMETHOD 2:60% Solvent B in 70 minutesTFA; C4 column0.325 mL / minute flow rateMETHOD 3:15-50% Solvent B in 70 minutesHFBA; C4 column
[0162]For gradients over Microbore LC columns a 50 μL / minute flow rate was used unless otherwise noted.
METHOD 4:60% Solvent B in 70 minutesTFA; C4 columnMETHOD 5:30% Solvent B in 60 m...
example 2
Protein Sequencing
[0163]Purified peptides were reduced with 10 mM dithiothreitol and alkylated with 4-vinylpyridine. The reduced and alkylated peptide was then isolated from the chemicals on a Magic 2002 Microbore LC using reversed phase chromatography. Protein sequencing was performed on an Applied Biosystems Procise 494 protein sequencer utilizing the Edman degradation reaction. Isolated peptides were pipetted onto a pre-filtered glass fiber filter from Applied Biosystems containing 100 mM Biobrene. The filter was dried and the cartridge was re-assembled and placed onto the Procise 494. N-terminal sequencing was determined with the pulsed-liquid method standard with the Procise 494. Data were collected and analyzed on the Model 610A data analysis program from Applied Biosystems.
example 3
Full Length Peptide Sequencing
[0164]Purified peptides were reduced and alkylated as mentioned previously. The peptides were then digested with GluC and LysC endoproteinases from Boehringer-Mannheim. These endoproteinases cleave on the C-terminal side of glutamic acid and lysine respectively in the peptide. Peptide fragments were collected on the Magic 2002 Microbore LC using reversed phase chromatography. Each individual peptide was sequenced as described previously until a full-length sequence map was obtained.
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