Claims
- 1. An isolated, modified IMPDH polypeptide comprising an oligo-peptide domain substituted for a subdomain of a wild-type IMPDH polypeptide, the substitution resulting in a modified IMPDH polypeptide, which is shorter in length compared to the wild-type IMPDH polypeptide.
- 2. The modified IMPDH polypeptide of claim 1, wherein the wild type IMPDH polypeptide is type I or type II IMPDH.
- 3. The modified IMPDH polypeptide of claim 1, further comprising a first IMPDH catalytic core domain and a second IMPDH catalytic core domain.
- 4. The isolated, modified IMPDH polypeptide of claim 3, wherein the oligo-peptide domain is located between the first and the second IMPDH catalytic core domains.
- 5. The isolated, modified IMPDH polypeptide of claim 3, wherein the first IMPDH catalytic core domain is located N-terminal to the second IMPDH catalytic core domain.
- 6. The modified IMPDH polypeptide of claim 1, wherein the oligo-peptide domain comprises a tri-peptide.
- 7. The modified IMPDH polypeptide of claim 1, wherein the oligo-peptide domain comprises a tetra-peptide.
- 8. The modified IMPDH polypeptide of claim 1 having the amino acid sequence as shown in any one of SEQ ID NOS.: 20-39.
- 9. The modified IMPDH polypeptide of claim 6, wherein the tri-peptide has an amino acid sequence as shown in any one of SEQ ID NOS.: 1-10.
- 10. The modified IMPDH polypeptide of claim 7, wherein the tetra-peptide has an amino acid sequence as shown in any one of SEQ ID NOS.:11-19.
- 11. The isolated, modified IMPDH polypeptide of claim 6, wherein the first amino acid position of the tri-peptide sequence is selected from the group consisting of aspartic acid, threonine, serine, or glycine, lysine, isoleucine and alanine.
- 12. The isolated modified IMPDH polypeptide of claim 6, wherein the second amino acid position of the tri-peptide sequence is selected from the group consisting of lysine, proline, alanine, valine, leucine, glycine and serine.
- 13. The isolated modified IMPDH polypeptide of claim 6, wherein the third amino acid position of the tri-peptide sequence is selected from the group consisting of tyrosine, serine, threonine, glycine, phenylalanine, isoleucine, histidine, and aspartic acid.
- 14. The isolated modified IMPDH polypeptide of claim 7, wherein the first amino acid position of the substitute tetra-peptide sequence is selected from the group consisting of glycine, glutamine, asparagine, serine, threonine, tyrosine, and alanine.
- 15. The isolated modified IMPDH polypeptide of claim 7, wherein the second amino acid position of the substitute tetra-peptide sequence is selected from the group consisting of serine, glycine, pro line, iso leucine, and arginine.
- 16. The isolated modified IMPDH polypeptide of claim 7, wherein the third amino acid position of the substitute tetra-peptide sequence is selected from the group consisting of serine, glutamine, threonine, tyrosine, isoleucine, proline, and arginine.
- 17. The isolated modified IMPDH polypeptide of claim 7, wherein the fourth amino acid position of the substitute tetra-peptide sequence is selected from the group consisting of tryptophan, proline, leucine, serine, glutamine, threonine, and tyrosine.
- 18. A protein multimer, comprising between 1 and 8 modified IMPDH polypeptides in association with each other, wherein the modified IMPDH polypeptides each comprise an oligo-peptide domain substituted for a subdomain of a wild-type IMPDH polypeptide resulting in the modified IMPDH polypeptide, which is shorter in length compared to the wild-type IMPDH polypeptide.
- 19. The protein multimer of claim 18 which is a dimer.
- 20. The protein multimer of claim 18 which is a tetramer.
- 21. The protein multimer of claim 18 which is an octamer.
- 22. A nucleic acid molecule comprising a polynucleotide sequence which encodes any one of the modified IMPDH polypeptides of claim 8.
- 23. The nucleic acid molecule of claim 22 which is RNA.
- 24. The nucleic acid molecule of claim 22 which is DNA.
- 25. A nucleic acid molecule comprising a polynucleotide sequence which is complementary to the polynucleotide sequence of claim 24.
- 26. The nucleic acid molecule of claim 22, 23, 24 or 25 which is labeled with a detectable marker.
- 27. The nucleic acid molecule of claim 26, wherein the detectable marker is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, and an enzyme.
- 28. A vector comprising a polynucleotide sequence which encodes any one of the modified IMPDH polypeptides of claim 8.
- 29. A host-vector system comprising the vector of claim 28 in a suitable host cell.
- 30. The host-vector system of claim 29, wherein the suitable host cell is from an organism which is selected from the group consisting of bacteria, yeast, mammals, insects, and plants.
- 31. A method for producing a modified IMPDH polypeptide comprising:
a) culturing the host-vector system of claim 30 under suitable conditions so as to produce the modified IMPDH polypeptide; and b) recovering the modified IMPDH polypeptide so produced.
- 32. The modified IMPDH polypeptide produced by the method of claim 31.
- 33. A monoclonal antibody reactive with the modified IMPDH polypeptide of claim 1 or 32.
- 34. The monoclonal antibody of claim 33 which is labeled with a detectable marker.
- 35. The monoclonal antibody of claim 34, wherein the detectable marker is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, and an enzyme.
- 36. A method for identifying an agent of interest that inhibits the activity of a protein multimer comprising modified IMPDH polypeptides, the method comprising:
a) contacting the protein multimer with inosine-5′-monophosphate, nicotinamide adenine dinucleotide, and the agent of interest; and b) detecting the level of the reduced form of inosine-5′-monophosphate or nicotinamide adenine dinucleotide which is generated, whereby a low level of the reduced form of inosine-5′-monophosphate or nicotinamide adenine dinucleotide generated indicates that the agent of interest inhibits IMPDH activity.
- 37. The method according to claim 36 which comprises contacting a plurality of substantially identical samples each separately with a different agent of interest.
- 38. The method of claim 37, wherein the plurality of samples comprises more than about 104 samples.
- 39. The method of claim 37, wherein the plurality of samples comprises more than about 105 samples.
- 40. The method of claim 37, wherein the plurality of samples comprises more than about 106 samples.
- 41. The method of claim 40, wherein the plurality of substantially identical samples are each contacted essentially simultaneously with a different agent of interest.
- 42. A method for improving resolution of an X-ray crystal structure of an IMPDH polypeptide or IMPDH polypeptide complex comprising removing a subdomain of the IMPDH polypeptide thereby improving the resolution of the X-ray crystal structure of the IMPDH polypeptide or IMPDH polypeptide.
- 43. The method of claim 42, wherein the IMPDH polypeptide has the amino acid sequence of any one of SEQ ID NOs:20-39.
- 44. The method of claim 42, wherein the protein or protein complex is complexed with a compound.
- 45. The method of claim 44, wherein the compound is an inhibitor.
- 46. The method of claim 45, wherein the inhibitor is MPA.
- 47. The method of claim 42, wherein the subdomain of the IMPDH polypeptide is replaced with a relatively shorter peptide fragment.
- 48. A method for improving resolution of an X-ray crystal structure of an IMPDH polypeptide comprising reducing the length of the IMPDH polypeptide thereby improving the resolution of the X-ray crystal structure of the IMPDH polypeptide.
- 49. A method for improving resolution of an X-ray crystal structure of an IMPDH polypeptide comprising substituting amino acids 111-243 of any one of SEQ ID NOS: 48, 49, 62, 63, 64, or 65 with a tri-peptide thereby improving the resolution of the X-ray crystal structure of the IMPDH polypeptide.
- 50. The method of claim 49, where in the tri-peptide has an amino acid sequence as shown in any one of SEQ ID NOS.:1-10.
- 51. A method for improving resolution of an X-ray crystal structure of an IMPDH polypeptide comprising substituting amino acids 111-243 of any one of SEQ ID NOS: 48, 49, 62, 63, 64, or 65 with a tetra-peptide thereby improving the resolution of the X-ray crystal structure of the IMPDH polypeptide.
- 52. The method of claim 51, wherein the tetra-peptide has an amino acid sequence as shown in SEQ ID NOs: 11-19.
- 53. A modified IMPDH polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOS.:20-39.
- 54. An isolated nucleic acid molecule comprising the nucleic acid sequence as shown in any one of SEQ ID NOS.:40-47.
- 55. A modified IMPDH polypeptide, wherein amino acids 111-243 as shown in any one of SEQ ID NOS:48, 49, 62, 63, 64, or 65 are replaced with a tripeptide.
- 56. A modifified IMPDH polypeptide, wherein amino acids 111-243 as shown in any one of SEQ ID NOS:48, 49, 62, 63, 64, or 65 are replaced with a tetrapeptide.
Parent Case Info
[0001] This application claims the benefit of the filing date of U.S. Ser. No. 60/203,448, filed May 10, 2000. The content of the foregoing application in its entirety is incorporated by reference herein.
[0002] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
Provisional Applications (1)
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Number |
Date |
Country |
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60203448 |
May 2000 |
US |