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Details for Patent: 6,428,954

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Details for Patent: 6,428,954

Title: Method for identifying active domains and amino acid residues in polypeptides and hormone variants
Abstract:The invention provides methods for the systematic analysis of the structure and function of polypeptides by identifying active domains which influence the activity of the polypeptide with a target substance. Such active domains are determined by substituting selected amino acid segments of the polypeptide with an analogous polypeptide segment from an analog to the polypeptide. The analog has a different activity with the target substance as compared to the parent polypeptide. The activities of the segment-substituted polypeptides are compared to the same activity for the parent polypeptide for the target. A comparison of such activities provides an indication of the location of the active domain in the parent polypeptide. The invention also provides methods for identifying the active amino acid residues within the active domain of the parent polypeptide. The method comprises substituting a scanning amino acid for one of the amino acid residues within the active domain of the parent polypeptide and assaying the residue-substituted polypeptide so formed with a target substance. The invention further provides polypeptide variants comprising segment-substituted and residue-substituted growth hormones, prolactens and placental lactogens.
Inventor(s): Wells; James A. (Burlingame, CA), Cunningham; Brian C. (Piedmont, CA)
Assignee: Genentech, Inc. (South San Francisco, CA)
Filing Date:Jun 06, 1995
Application Number:08/483,039
Claims:1. A method for identifying at least a first unknown active domain in a region of known amino acid sequence of a parent polypeptide, which parent polypeptide has been cloned and has a pre-identified biological activity, said active domain capable of interacting with a first target when said parent polypeptide is in its native-folded form, which interaction is responsible for said biological activity, said method comprising: (a) comparing amino acid sequence or polypeptide structure in the region of known amino acid sequence of the parent polypeptide with amino acid sequence or polypeptide structure in a region of known amino acid sequence of an analog polypeptide to said parent polypeptide, said analog having at least 15% sequence homology with said parent polypeptide or .alpha.-carbon coordinates within about 2 to about 3.5 .ANG. of parent polypeptide .alpha.-carbon coordinates for about 60% or more of the analog sequence, wherein any interaction of said analog with said first target is different from the interaction of said parent polypeptide with said first target; (b) substituting DNA encoding a first analogous polypeptide segment from said analog into DNA encoding substantially the full-length parent polypeptide and expressing a first segment-substituted polypeptide; (c) contacting said first segment-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said segment-substituted polypeptide; (d) repeating steps (b) and (c) using a second analogous polypeptide segment from an analog to said parent polypeptide to form at least a second segment-substituted polypeptide containing said second analogous polypeptide segment, which is different from said first analogous polypeptide segment; and (e) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and said first and second segment-substituted polypeptides as an indication of the location of said first unknown active domain in said parent polypeptide.

2. The method of claim 1 wherein said first unknown active domain comprises at least two discontinuous amino acid segments in the primary amino acid sequence of said parent polypeptide.

3. The method of claim 1 wherein at least a first selected polypeptide segment of said parent polypeptide replaced by said first analogous polypeptide segment of said analog contains at least one amino acid residue located on the surface of the native-folded form of said parent polypeptide.

4. The method of claim 3 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of substantially all of the amino acid residues on said surface of said parent polypeptide.

5. The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous polypeptide segments covering about 15-100% of the amino acid sequence of said parent polypeptide.

6. The method of claim 1 further comprising repeating steps (b) and (c) to form a plurality of segment-substituted polypeptides that, collectively, contain substitutions of analogous polypeptide segments covering about 60-100% of the amino acid sequence of said parent polypeptide.

7. The method of claim 1 further comprising identifying a second unknown active domain of said parent polypeptide, said second active domain interacting with a second target, said method comprising repeating steps (b) through (e) with said second target.

8. The method of claim 1 further comprising identifying at least a first active amino acid residue within said first active domain, said method comprising: (f) substituting a scanning amino acid for a different first amino acid residue within said first active domain to form a first residue-substituted polypeptide; (g) contacting said first residue-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said first residue-substituted polypeptide; (h) repeating steps (f) and (g) to substitute a scanning amino acid for at least a second amino acid residue within said first active domain to form at least a second residue-substituted polypeptide; and (i) comparing the difference, if any, between the activity to relative to said first target of said parent polypeptide and each of said first and second residue-substituted polypeptides as an indication of the location of said first active amino acid residue in said first active domain.

9. The method of claim 8 further comprising repeating steps (b) through (i) with a second target to identify a second active domain and at least one active amino acid residue within said second active domain.

10. The method of claim 9 further comprising the step of substituting an active amino acid residue in said first active domain with different amino acid to produce a polypeptide variant having a modified interaction with said first target but which retains substantially all of the interaction of said parent polypeptide with said second target.

11. The method of claim 10 further comprising the step of substituting an active amino acid residue in said second active domain with a different amino acid to produce a polypeptide variant having modified interaction with said second target but which retains the interaction of said parent polypeptide with said first target.

12. The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least said one common active amino acid residue with a different amino acid to produce a polypeptide variant having modified interactions with each of said first and said second targets.

13. The method of claim 9 wherein said first and said second active domains have at least one common active amino acid residue, said method further comprising substituting at least one amino acid residue in said first active domain, other than said at least one common active amino acid residue, with a different amino acid to produce a polypeptide variant having a modified interaction with said first target.

14. A method for identifying at least one active amino acid residue in a parent polypeptide, which parent polypeptide has been cloned and has a pre-identified biological activity resulting from an interaction with a first target, comprising: (a) substituting DNA encoding a scanning amino acid for DNA encoding a first amino acid residue at residue number N within DNA encoding substantially the full-length parent polypeptide and expressing an N-substituting polypeptide; (b) substituting DNA encoding a scanning amino acid for each of the amino acid residues at residue numbers N+1 and N-1 to said first residue within DNA encoding substantially the full-length parent polypeptide and expressing N+1- and N-1-substituted polypeptides, respectively; (c) contacting each of said substituted polypeptides expressed in steps (a) and (b) with said first target to determine the interaction, if any, between said first target and said substituted polypeptides; and (d) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and said substituted polypeptides as an indication of the location of said active amino acid residue.

15. The method of claim 14 wherein residue N is a suspected active amino acid residue and steps (b) through (d) are repeated until at least four consecutive residues are identified wherein substitution of a scanning amino acid at each of said four conservative residues has less than a two-fold effect on activity with said first target as compared to said parent polypeptide.

16. The method of claim 14 wherein said parent polypeptide is selected from the group consisting of human growth hormone, human placental lactogen, and human prolactin.

17. The method of claim 14 wherein at least one of said scanning amino acids is an isosteric amino acid.

18. The method of claim 14 wherein the same scanning amino acid is employed in steps (a) and (b), and said scanning amino acid is a neutral amino acid.

19. The method of claim 18 wherein said neutral amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.

20. The method of claim 19 wherein said scanning amino acid is alanine.

21. The method of claim 14 wherein said parent polypeptide is hormone and said activity is measured in an in vitro assay using a soluble hormone receptor.

22. The method of claim 21 wherein said hormone is human growth hormone and said soluble hormone receptor is shGHr.

23. The method of claim 21 wherein said hormone is human growth hormone and said soluble hormone receptor is shPRLr.

24. The method of claim 14 wherein said interaction between said first target and said parent polypeptide involves either binding or catalytic interaction of said parent polypeptide with said first target.

25. The method of claim 24 wherein the activity between said first target and any of said substituted polypeptides is increased greater than two-fold as compared to said parent polypeptide.

26. The method of claim 24 wherein the activity between said first target and any of said substituted polypeptides is decreased greater than two-fold as compared to said parent polypeptide.

27. The method of claim 1 wherein said parent polypeptide is selected from the group consisting of growth consisting of growth hormone, prolactin, placental lactogen, .alpha.-interferon, .gamma.interferon, TGF-.beta..sub.1, EGF, IGF-1, GM-CSF, TNF, tissue plasminogen activator, and CD-4 receptor.

28. The method of claim 27 wherein said parent polypeptide is selected from the group consisting of human growth hormone , human placental lactogen, and human prolactin.

29. The method of claim 1 wherein said activity is measured in an in vitro assay.

30. The method of claim 29 wherein said parent polypeptide is a hormone and said activity is measured in an in vitro assay using a soluble hormone receptor.

31. The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shGHr.

32. The method of claim 30 wherein said hormone is human growth hormone and said soluble hormone receptor is shPRLr.

33. The method of claim 1 wherein said interaction between said first target and said parent polypeptide involves either binding or catalytic interaction of said parent polypeptide with said first target.

34. The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is increased greater than two-fold as compared to said parent polypeptide.

35. The method of claim 33 wherein the activity between said first target and any of said substituted polypeptides is decreased greater than two-fold as compared to said parent polypeptide.

36. The method of claim 1, wherein said analog has at least 15% amino acid sequence homology with said parent polypeptide.

37. The method of claim 1, wherein said analog is naturally occurring.

38. The method of claim 1, wherein said analog is a tertiary analog.

39. The method of claim 1 wherein said parent polypeptide is human growth hormone and said analog is selected from the group consisting of human placental lactogen, porcine growth hormone, and human prolactin.

40. The method of claim 1 wherein the biological activity of the parent polypeptide is of clinical utility.

41. The method of claim 1 wherein said parent polypeptide is selected from the group consisting of a hormone, enzyme, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.

42. The method of claim 1 wherein said first target is selected from the group consisting of a hormone, enzyme, antibody, antigen, receptor, enzyme substrate, binding protein, and enzyme inhibitor.

43. The method of claim 1 wherein said analog does not substantially interact with said first target. binding

44. The method of claim 8 wherein at least one of said scanning amino acids is an isosteric amino acid.

45. The method of claim 8 wherein the same scanning amino acid is employed in steps (f) and (h), and said scanning amino acid is a neutral amino acid.

46. The method of claim 45 wherein said neutral amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.

47. The method of claim 46 wherein said scanning amino acid is alanine.

48. The method of claim 1 wherein said parent polypeptide is naturally occurring.

49. The method of claim 14 wherein said parent polypeptide is naturally occurring.
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