Butyrylcholinesterase variants and methods of use
AssigneesBoard of Regents of the University of Nebraska, Applied Molecular Evolution, Inc.
InventorsOksana Lockridge, Jeffry D. Watkins
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Abstract
The invention provides four butyrylcholinesterase variants having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acids. The invention also provides libraries comprising butyrylcholinesterase variants having at least one amino acid alteration in one or more regions of butyrylcholinesterase and further having at least one butyrylcholinesterase variant exhibiting enhanced cocaine hydrolysis activity compared to butyrylcholinesterase. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition.
Claims39
A butyrylcholinesterase variant comprising substantially the same amino acid sequence shown as SEQ ID NO:
- 2, or functional fragment thereof.
A nucleic acid encoding a butyrylcholinesterase variant comprising substantially the same nucleic acid sequence shown as SEQ ID NO:
- 1, or fragment thereof.
A butyrylcholinesterase variant comprising substantially the same amino acid sequence shown as SEQ ID NO:
- 4, or functional fragment thereof.
A nucleic acid encoding a butyrylcholinesterase variant comprising substantially the same nucleic acid sequence shown as SEQ ID NO:
- 3, or fragment thereof.
A butyrylcholinesterase variant comprising substantially the same amino acid sequence shown as SEQ ID NO:
- 6, or functional fragment thereof.
A nucleic acid encoding a butyrylcholinesterase variant comprising substantially the same nucleic acid sequence shown as SEQ ID NO:
- 5, or fragment thereof.
A butyrylcholinesterase variant comprising substantially the same amino acid sequence shown as SEQ ID NO:
- 8, or functional fragment thereof.
A nucleic acid encoding a butyrylcholinesterase variant comprising substantially the same nucleic acid sequence shown as SEQ ID NO:
- 7, or fragment thereof.
A library comprising butyrylcholinesterase variants having at least one amino acid alteration in one or more regions of butyrylcholinesterase corresponding to amino acid positions 68-82 (SEQ ID NO:
- 9), 110-121 (SEQ ID NO:
- 10), 194-201 (SEQ ID NO:
- 11), 224-234 (SEQ ID NO:
- 12), 277-289 (SEQ ID NO:
- 13), 327-332 (SEQ ID NO:
- 14) or 429-442 (SEQ ID NO:
- 15) of butyrylcholinesterase or functional fragment thereof, said library having at least one butyrylcholinesterase variant exhibiting enhanced cocaine hydrolysis activity compared to butyrylcholinesterase, with the proviso that a butyrylcholinesterase variant having a single amino acid alteration is not the human butyrylcholinesterase having Y at position 328.
A library comprising nucleic acids encoding butyrylcholinesterase variants, each nucleic acid having at least one codon encoding at least one amino acid alteration in one or more regions of butyrylcholinesterase corresponding to amino acid positions 68-82 (SEQ ID NO:
- 9), 110-121 (SEQ ID NO:
- 10), 194-201 (SEQ ID NO:
- 11), 224-234 (SEQ ID NO:
- 12), 277-289 (SEQ ID NO:
- 13), 327-332 (SEQ ID NO:
- 14) or 429-442 (SEQ ID NO:
- 15) of butyrylcholinesterase, or functional fragment thereof, at least one of said nucleic acids encoding a butyrylcholinesterase variant having enhanced cocaine hydrolysis activity compared to butyrylcholinesterase, with the proviso that a butyrylcholinesterase variant having a single amino acid alteration is not the human butyrylcholinesterase having Y at position 328.
A method of hydrolyzing a cocaine-based butyrylcholinesterase substrate comprising contacting said butyrylcholinesterase substrate with a butyrylcholinesterase variant shown as SEQ ID NO:
- 2, or functional fragment thereof, under conditions that allow hydrolysis of cocaine into metabolites, wherein said butyrylcholinesterase variant exhibits a five-fold or more increase in cocaine hydrolysis activity compared to butyrylcholinesterase.
A method of treating a cocaine-induced condition comprising administering to an individual an effective amount of a butyrylcholinesterase variant shown as SEQ ID NO:
- 2, or functional fragment thereof, exhibiting increased cocaine hydrolysis activity compared to butyrylcholinesterase.
A method of hydrolyzing a cocaine-based butyrylcholinesterase substrate comprising contacting said butyrylcholinesterase substrate with a butyrylcholinesterase variant selected from the group consisting of SEQ ID NO:
- 4, SEQ ID NO:
- 6 and SEQ ID NO:
- 8, or functional fragment thereof, under conditions that allow hydrolysis of cocaine into metabolites, wherein said butyrylcholinesterase variant exhibits a two-fold or more increase in cocaine hydrolysis activity compared to butyrylcholinesterase.
A method of treating a cocaine-induced condition comprising administering to an individual an effective amount of a butyrylcholinesterase variant selected from the group consisting of SEQ ID NO:
- 4, SEQ ID NO:
- 6 and SEQ ID NO:
- 8, or functional fragment thereof, exhibiting increased cocaine hydrolysis activity compared to butyrylcholinesterase.
Dependencies are inferred from the claim text.