Enzymes having thermostable phosphatase activity and methods of use thereof
OwnerJay M. Short, Eric J. Mathur +2inventors
InventorsJay M. Short, Eric J. Mathur, Edd Lee, Edward Bylina
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Abstract
The invention relates to thermostable phosphatases and to polynucleotides encoding the thermostable phosphatases. In addition methods of designing new phosphatases and methods of use thereof are also provided. The thermostable phosphatases have increased activity and stability at increased pH and temperature.
Claims92
An isolated nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and variants thereof having at least about 50% identity to SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and encoding a polypeptide having phosphatase activity.
An isolated nucleic acid comprising at least 10 consecutive bases of a sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, sequences substantially identical thereto, and sequences complementary thereto.
An isolated nucleic acid encoding a polypeptide having a sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
An isolated nucleic acid encoding a polypeptide comprising at least 10 consecutive amino acids of a polypeptide having a sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A purified polypeptide having a sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A purified antibody that specifically binds to a polypeptide comprising a sequence selected from the group consisting of SEQ IDNOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A purified antibody that specifically binds to a polypeptide having at least 10 consecutive amino acids of the polypeptides selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A method of producing a polypeptide having a sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.
A method of producing a polypeptide comprising at least 10 amino acids of a sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide, operably linked to a promoter, into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.
A method of generating a variant comprising:
- obtaining a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, sequences substantially identical thereto, sequences complementary thereto, fragments comprising at least 30 consecutive nucleotides thereof, and fragments comprising at least 30 consecutive nucleotides of the sequences complementary to SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53;
- and
- modifying one or more nucleotides in said sequence to another nucleotide, deleting one or more nucleotides in said sequence, or adding one or more nucleotides to said sequence.
A computer readable medium having stored thereon a nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto.
A method for comparing a first sequence to a reference sequence wherein said first sequence is a nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto comprising:
- reading the first sequence and the reference sequence through use of a computer program which compares sequences;
- and
- determining differences between the first sequence and the reference sequence with the computer program.
A method for identifying a feature in a sequence wherein the sequence is selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 3 8, 39, 40, 41, 43, 45, 47, 49, 51, 53, sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto comprising:
- reading the sequence through the use of a computer program which identifies features in sequences;
- and
- identifying features in the sequences with the computer program.
A method of catalyzing the hydrolysis of phosphates comprising contacting a sample containing phosphatase with a polypeptide selected from the group consisting of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences having at least 50% homology and having phosphatase enzyme activity under conditions which facilitate the hydrolysis of phosphates.
An assay for identifying functional polypeptide fragments or variants encoded by fragments of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and sequences substantially identical thereto, which retain the enzymatic function of the polypeptides of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto, said assay comprising:
- contacting the polypeptide of SEQ ID NOS:
- 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 43, 44, 46, 47, 48, 50, 52, 54, and sequences substantially identical thereto, or polypeptide fragment or variant encoded by SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, with a substrate molecule under conditions which allow said polypeptide or fragment or variant to function, and
- detecting either a decrease in the level of substrate or an increase in the level of the specific reaction product of the reaction between said polypeptide and substrate, wherein a decrease in the level of substrate or an increase in the level of the reaction product is indicative of a functional polypeptide or fragment or variant.
A nucleic acid probe comprising an oligonucleotide from about 10 to 50 nucleotides in length and having an area of at least 10 contiguous nucleotides that is at least 50% complementary to a nucleic acid target region of the nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 90% complementary to a nucleic acid target region of the nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 95% complementary to a nucleic acid target region of the nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 97% complementary to a nucleic acid target region of the nucleic acid sequence selected from the group consisting of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51, 53, and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A polynucleotide probe for isolation or identification of phosphatase genes having a sequence which is the same as or fully complementary to at least a portion of SEQ ID NOS:
- 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, 40, 41, 43, 45, 47, 49, 51 or 53.
Dependencies are inferred from the claim text.