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Details for Patent: 5,834,250

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Details for Patent: 5,834,250

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, prolactins 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 30, 1997
Application Number:08/903,398
Claims:1. A method for identifying at least one active amino acid residue in a parent polypeptide which parent polypeptide has an activity resulting from an interaction with a target, said method comprising:

(a) substituting a scanning amino acid for a first amino acid residue at residue number N within said parent polypeptide to form an N-substituted polypeptide;

(b) substituting a scanning amino acid for each of the amino acid residues at residue numbers N+1 and N-1 to said first residue to form respectively N+1- and N-1-substituted polypeptides;

(c) contacting each of said substituted polypeptides with a target to determine the interaction, if any, between said target and said substituted polypeptides;

(d) comparing the difference, if any, between the activity of the parent polypeptide and said substituted polypeptides with said target as an indication of the location of said active amino acid residue in said parent polypeptide;

(e) repeating steps (b) through (d) for increasing residue numbers if said activity difference between said target and said N+1 substituted polypeptide is greater than two-fold and for decreasing residue numbers if said activity difference between said target and said N-1 substituted polypeptide is greater than two-fold.

2. The method of claim 1 wherein steps (b) through (d) are repeated until at least four substituted polypeptides containing the substitution of a scanning amino acid at four consecutive residues are identified having less than a two-fold activity difference as compared to said parent polypeptide.

3. A method for identifying at least one active amino acid residue in a polypeptide, comprising the steps of:

(a) separately substituting a single scanning amino acid for at least three amino acid residues in at least one domain of a polypeptide of known amino acid sequence, wherein the polypeptide has a known interaction with a target, to form at least three corresponding substituted polypeptides, each substituted polypeptide containing one scanning amino acid;

(b) contacting the corresponding substituted polypeptides with the target to determine an interaction, if any, between the target and the corresponding substituted polypeptides; and

(c) comparing the difference, if any, between the interaction of the target with the polypeptide and The interaction of the target with the corresponding substituted polypeptides as an indication of the location of said active amino acid residue in said polypeptide.

4. The method of claim 3, comprising separately substituting a single scanning amino acid for at least 12 amino acid residues in at least one domain of the polypeptide.

5. The method of claim 3, comprising separately substituting a single scanning amino acid for at least 18 amino acid residues in at least one domain of the polypeptide.

6. The method of claim 5, comprising separately substituting a single scanning amino acid for at least 18-25 amino acid residues in at least one domain of the polypeptide.

7. The method of claim 3, comprising separately substituting a single scanning amino acid for each amino acid residue in at least one domain of the polypeptide.

8. The method of claim 3, wherein the domain comprises 3-30 amino acid residues.

9. The method of claim 8, wherein the domain comprises 3-15 amino acid residues.

10. The method of claim 7, wherein the domain comprises at least 12 amino acid residues.

11. The method of claim 3, comprising separately substituting the scanning amino acid in two or more domains of a polypeptide.

12. The method of claim 3, wherein the single scanning amino acid is a neutral amino acid.

13. The method of claim 12, wherein the neutral amino acid is alanine.

14. The method of claim 3, wherein the known interaction is the binding of the target to the polypeptide.

15. The method of claim 3, consisting of steps (a), (b), (c).

16. A method for identifying at least one active amino acid residue in a polypeptide, comprising the steps of:

a) separately substituting a single scanning amino acid for each amino acid in a polypeptide of known amino acid sequence containing 12-50 amino acid residues, wherein the polypeptide has a known interaction with a target, to form corresponding substituted polypeptides;

(b) contacting the corresponding substituted polypeptides with the target to determine an interaction, if any, between the target and the corresponding substituted polypeptides; and

(c) comparing the difference, if any, between the interaction of the target with the polypeptide and the interaction of the target with the corresponding substituted polypeptides as an indication of the location of said active amino acid residue in said polypeptide.

17. The method of claim 16, wherein the polypeptide comprises about 18-50 amino acid residues.

18. The method of claim 16, wherein the single scanning amino acid is a neutral amino acid.

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

20. The method of claim 19, wherein the neutral amino acid is alanine.

21. The method of claim 16, wherein the known interaction is the binding of the target to the polypeptide.

22. The method of claim 16, consisting of steps (a), (b), (c).

23. The method of claim 1, wherein the scanning amino acid is selected from the group consisting of Ser, Gly, Gln, Asp, Asn, Glu, Met, lle, Pro, Arg, Thr, Lys, Val, Tyr and Phe.

24. The method of claim 1, wherein the scanning amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.

25. The method of claim 1, wherein the scanning amino acid is alanine.

26. The method of claim 1, wherein the scanning amino acid is cysteine.

27. The method of claim 3, wherein the scanning amino acid is selected from the group consisting of Ser, Gly, Gln, Asp, Asn, Glu, Met, lle, Pro, Arg, Thr, Lys, Val, Tyr and Phe.

28. The method of claim 3, wherein the scanning amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.

29. The method of claim 3, wherein the scanning amino acid is cysteine.

30. The method of claim 16, wherein the scanning amino acid is selected from the group consisting of Ser, Gly, Gln, Asp, Asn, Glu, Met, lle, Pro, Arg, Thr, Lys, Val, Tyr and Phe.

31. The method of claim 16, wherein the scanning amino acid is cysteine.
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