Molecules and methods for iterative polypeptide analysis and processing
AssigneeWashington University
InventorsJames J. Havranek, Benjamin Borgo
Life of this patent
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Abstract
Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.
Claims24
1-22. (canceled)
An isolated, synthetic, or recombinant Edman degradation enzyme comprising an amino acid sequence having a glycine residue at a position corresponding to position 25 of wild-type Trypanosoma cruzi cruzian;
- a serine residue at a position corresponding to position 65;
- a cysteine residue at a position corresponding to position 138;
- and a tryptophan residue at a position corresponding to position 160;
- wherein the remaining amino acid sequence of the Edman degradation enzyme comprises a sequence having at least about 80% sequence identity to the amino acid sequence of wild-type Trypanosoma cruzi cruzian.
A method for sequencing a polypeptide, the method comprising:
- (a) contacting the polypeptide with one or more fluorescently labeled N-terminal amino acid binding proteins (NAABs);
- (b) detecting fluorescence of a NAAB bound to an N-terminal amino acid of the polypeptide;
- (c) identifying the N-terminal amino acid of the polypeptide based on the fluorescence detected;
- (d) removing the NAAB from the polypeptide;
- (e) optionally repeating steps (a) through (d);
- (f) cleaving the N-terminal amino acid of the polypeptide via Edman degradation;
- and
- (g) repeating steps (a) through (f) one or more times.
39-42. (canceled)
44-45. (canceled)
A method for cleaving the N-terminal amino group of a polypeptide, the method comprising:
- (a) reacting the N-terminal amino acid of the polypeptide with phenyl isothiocyanate (PITC) to generate a PITC-derivatized N-terminal amino acid;
- and
- (b) cleaving the modified N-terminal amino group with an Edman degradation enzyme.
49-83. (canceled)
Dependencies are inferred from the claim text.