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Details for Patent: 6,013,478

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Details for Patent: 6,013,478

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 24, 1998
Application Number:09/104,036
Claims:1. A method for producing a product polypeptide, comprising the steps of:

(1) culturing a host cell transformed with a replicable expression vector, the replicable expression vector comprising DNA encoding a product polypeptide operably linked to a control sequence capable of effecting expression of the product polypeptide in the host cell;

wherein the DNA encoding the product polypeptide has been obtained by a method comprising the steps of:

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

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

(c) comparing the difference, between the interaction of the target with the polypeptide and the interaction of the target with the corresponding substituted polypeptides as an indication of location of active amino acid residue in the polypeptide of known amino acid sequence;

(d) substituting a different amino acid for the scanning amino acid in at least one of the corresponding substituted polypeptides to form modified polypeptides;

(e) selecting one of the modified polypeptides as the product polypeptide and cloning DNA encoding the product polypeptide into the replicable expression vector; and

(2) recovering the expressed product polypeptide.

2. The method of claim 1, comprising separately substituting a single scanning amino acid for at least 12 amino acid residues in at least one domain of the polypeptide.

3. The method of claim 1, comprising separately substituting a single scanning amino acid for at least 18 amino acid residues in at least one domain of the polypeptide.

4. The method of claim 3, comprising separately substituting a single scanning amino acid for 18-25 amino acid residues in at least one domain of the polypeptide.

5. The method of claim 1, comprising separately substituting a single scanning amino acid for each amino acid residue in at least one domain of the polypeptide.

6. The method of claim 1, wherein the domain comprises 3-30 amino acid residues.

7. The method of claim 6, wherein the domain comprises 3-15 amino acid residues.

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

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

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

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

12. The method of claim 1, wherein the known interaction is the binding of the target to the polypeptide.

13. The method of claim 12, wherein the product polypeptide has at least a two-fold increase or decrease in binding interaction with the target relative to the polypeptide.

14. A product polypeptide produced by the method of claim 1.

15. A method for producing a product polypeptide, comprising the steps of:

(1) culturing a host cell transformed with a replicable expression vector, the replicable expression vector comprising DNA encoding a product polypeptide operably linked to a control sequence capable of effecting expression of the product polypeptide in the host cell;

wherein the DNA encoding the product polypeptide has been obtained by a method comprising the steps of:

(a) separately substituting a single scanning amino acid for each amino acid in a polypeptide 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 between the target and the corresponding substituted polypeptides; and

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

(d) substituting a different amino acid for the scanning amino acid in at least one of the corresponding substituted polypeptides to form modified polypeptides;

(e) selecting one of the modified polypeptides as the product polypeptide and cloning DNA encoding the product polypeptide into the replicable expression vector; and

(2) recovering the expressed product polypeptide.

16. The method of claim 15, wherein the polypeptide comprises about 18-50 amino acid residues.

17. The method of claim 15, wherein the scanning amino acid is selected from the group consisting of Ser, Gly, Gln, Asp, Asn, Glu, Met, Ile, Pro, Arg, Thr, Lys, Val, Tyr and Phe.

18. The method of claim 16, wherein the scanning amino acid is selected from the group consisting of alanine, serine, glycine, and cysteine.

19. The method of claim 15, wherein the scanning amino acid is cysteine.

20. The method of claim 15, wherein the scanning amino acid is alanine.

21. The method of claim 15, wherein the known interaction is the binding of the target to the polypeptide.

22. The method of claim 21, wherein the product polypeptide has at least a two-fold increase or decrease in binding interaction with the target relative to the polypeptide.

23. A product polypeptide produced by the method of claim 15.

24. A method for producing a product polypeptide, comprising the steps of:

(1) culturing a host cell transformed with a replicable expression vector, the replicable expression vector comprising DNA encoding a product polypeptide operably linked to a control sequence capable of effecting expression of the product polypeptide in the host cell;

wherein the DNA encoding the product polypeptide has been obtained by a method comprising the steps of:

(a) substituting a scanning amino acid for a first amino acid residue at residue number N within a polypeptide having a known activity resulting from an interaction with a target 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 the first residue to form respectively N+1- and N-1-substituted polypeptides;

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

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

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

(f) substituting a different amino acid for the scanning amino acid in at least one of the substituted polypeptides to form modified polypeptides;

(g) selecting one of the modified polypeptides as the product polypeptide and cloning DNA encoding the product polypeptide into the replicable expression vector; and

(2) recovering the expressed product polypeptide.

25. The method of claim 24, wherein the scanning amino acid is selected from the group consisting of Ser, Gly, Gln, Asp, Asn, Glu, Met, Ile, Pro, Arg, Thr, Lys, Val, Tyr and Phe.

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

27. The method of claim 24, wherein the scanning amino acid is alanine.

28. The method of claim 24, wherein the scanning amino acid is cysteine.

29. The method of claim 24, 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 polypeptide.

30. The method of claim 24, wherein the known interaction is the binding of the target to the polypeptide.

31. The method of claim 24, wherein the product polypeptide has at least a two-fold increase or decrease in binding interaction with the target relative to the polypeptide.

32. A product polypeptide produced by the method of claim 24.
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