Details for Patent: 6,780,613

Title:	Growth 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 07, 1995
Application Number:	08/479,884
Claims:	1. A DNA molecule comprising a nucleic acid sequence encoding a variant of a human growth hormone that binds to a target with an affinity different from the affinity of said human growth hormone for said target, wherein the amino acid sequence of said variant is not found in nature, and said variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of P2, T3, P5, S7, L9, N12, L15, R16, R19, E30, E33, S43, F44, Q46, N47, P48, Q49, T50, F54, S55, E56, S57, I58, P59, S62, N63, F66, Q68, K70, S71, L73, R77, L80, F97, A98, N99, S100, L101, V102, Y103, G104, D169, T175, R178, Q181, C182, R183, S184, V185, E186, G187, S188, and F191, numbered from the N-terminus of 191-amino acid human growth hormone, wherein said variant is not hGH-V. 2. The DNA molecule of claim 1, wherein said amino acid residue is selected from the group consisting of F97, S100, L101, V102, Y103, and T175, numbered from the N-terminus of 191-amino acid human growth hormone. 3. An expression vector comprising the DNA molecule of claim 2. 4. An expression host comprising an expression vector including the DNA molecule of claim 2. 5. An expression vector comprising the DNA molecule of claim 1. 6. An expression host comprising an expression vector including the DNA molecule of claim 1. 7. A method of producing a growth hormone variant comprising culturing the expression host of claim 6 and recovering said growth hormone variant from the culture. 8. The DNA molecule of claim 1 further comprising an amino acid substitution at amino acid residue G120. 9. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of P2, N12, L15, R16, and R19. 10. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of T3, P5, S7, and L9. 11. The DNA molecule of claim 1, wherein the variant comprises amino acid substitution at an amino acid residue selected from the group consisting of E30, E33, S43, F44, and Q46. 12. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of N47, P48, Q49, T50, and F54. 13. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S55, E56, S57, I58, and P59. 14. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S62, N63, E66, Q68, and K70. 15. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S71, L73, R77, L80, and F97. 16. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of A98, N99, S100, L101, and V102. 17. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of Y103, G104, D169, T175, and R178. 18. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of Q181, C182, R183, S184, and V185. 19. The DNA molecule of claim 1, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of E186, G187, S188, and F191. 20. A DNA molecule comprising a nucleic acid sequence encoding a variant of human growth hormone that binds to a target with an affinity different from the affinity of said human growth hormone for said target, wherein the amino acid sequence of said variant is not found in nature, and said variant consists of a set of amino acid substitutions selected from the group consisting of; N12H: F25L; D11A: M14V: H18Q: R19H: F25A: Q29K: E33R; N12R: M14V: L15V: R16L: R19Y: F25S: D26E: Q29S; E30Q: E33K; N12R: M14V: L15V: R16L: R19Y; Q22N: F25S: D26E: Q29S: E30Q: E33K; Q46H: N47D: P48S: Q49E; L52F; P48A: T50A: S51A: L52F; E56D: R64M; S57T: T60A: S62T: N63G: R64K: E65D: T67A: K70R; N72D: L73V; F54H: S55T: F56S: I58L: P59A: S62E: N63D: R64K: E66Q: T67A: K70M: S71N: N72Q: L73K: E74D; E88G: Q91Y: F92H: R94T: S59E; F97R: A98G: N99M: S100Q: L101D: V102A: Y103P: G104 E; N109D: V110D: D112H; Y111V: L131I: K115E: D116Q: E118K: E119R: G120L: Q122E: T123G: G126L: R127I: E129S; R127D: L128V: E129H: D130P: G131E: S132T: P133K: R134E: T135N; and R167K: D171H: T179V: Q181K, numbered from the N-terminus of 191-amino acid human growth hormone. 21. An expression vector comprising the DNA molecule of claim 20. 22. An expression host comprising an expression vector including the DNA molecule of claim 20. 23. A variant of a human growth hormone that binds to a target with an affinity different from the affinity of said human growth hormone for said target, wherein the amino acid sequence of said variant is not found in nature, and said variant consists of a set of amino acid substitutions selected from the group consisting of: N12H: F25L; D11A: M14V: H18Q: R19H: F25A: Q29K: E33R; N12R: M14V: L15V: R16L: R19Y: F25S: D26E: Q29S: E30Q: E33K; N12R: M14V: L15V: R16L: R19Y; Q22N: F25S: D26E: Q29S: E30Q: E33K; Q46H: N47D: P48S: Q49E: L52F; P48A: T50A: S51A: L52F; E56D: R64M; S57T: T60A: S62T: N63G: R64K: E65D: T67A: K70R: N72D: L73V; F54H: S55T: E56S: I58L: P59A: S62E: N63D: R64K: E66Q: T67A: K70M: S71N: N72Q: L73K: E74D; E88G: Q91Y: F92H: R94T: S95E; F97R: A98G: N99M: S100Q: L101D: V102A: Y103P: G104E; N109D: V110D: D112H; Y111V: L113I: K115E: D116Q: E118K: E119R: G120L: Q122E: T123G: G126L: R127I: E129S; R127D: L128V: E129H: D130P: G131E: S132T: P133K: R134E: T135N; and R167K: D171H: I179V: Q181K, numbered from the N-terminus of 191-amino acid human growth hormone. 24. A variant of a human growth hormone that binds to a target with an affinity different from the affinity of said human growth hormone for said target, wherein the amino acid sequence of said variant is not found in nature, and said variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of P2, T3, P5, S7, L9, N12, L15, R16, R19, E30, E33, S43, F44, Q46, N47, P48, Q49, T50, F54, S55, E56, S57, I58, P59, S62, N63, E66, Q68, K70, S71, L73, R77, L80, F97, A98, N99, S100, L101, V102, Y103, G104, D169, T175, R178, Q181, C182, R183, S184, V185, E186, G187, S188, and F191, numbered from the N-terminus of 191-amino acid human growth hormone, wherein said variant is not hGH-V. 25. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of P2, F54, E56, I58, Q68, Q69, L73, T175, R178, C182, S184, V185, E186, S188, and F191, numbered from the N-terminus of 191-amino acid human growth hormone. 26. The human growth hormone variant of claim 25 wherein said amino acid substitution is selected from the group consisting of P2A, F54G, F54E, F54M, F54A, F54R, F54Q, F54S, F54Y, F54W, F54L, F54I, F54V, E56G, E56M, E56F, E56A, E56R, E56Q, E56S, E56D, E56N, E56K, E56L, E56H, I58G, I58E, I58M, I58F, I58A, I58R, I58Q, I58S, I58V, I58T, Q68G, Q68E, Q68M, Q68F, Q68A, Q68R, Q68S, Q68H, Q68K, Q68D, Q68N, Q69A, L73A, T175G, T175E, T175M, T175F, T175R, T175Q, T175S, T175V, T175I, R178G, R178E, R178M, R178F, R178A, R178S, R178H, R178K, R178D, R178N, C182G, C182E, C182M, C182F, C182A, C182R, C182Q, C182S, S184A, V185G, V185E, V185M, V185F, V185A, V185R, V185Q, V185S, V815I, V185T, V185L, V185Y, V815W, E186A, S188A, and F191A. 27. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of N12, L15, R16, R19, S55, S57, P59, S62, N63, E66, K70, S71, C182, R183, and G187, numbered from the N-terminus of 191-amino acid human growth hormone. 28. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of P2, Q69, L73, S184, E186, S188, and F191, numbered from the N-terminus of 191 amino acid human growth hormone. 29. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of L73, E186, S188, and F191, numbered from the N-terminus of 191-amino acid human growth hormone. 30. The human growth hormone variant of claim 29 wherein said amino acid substitution is selected from the group consisting of L73A, E186A, S188A, and F191A. 31. The human growth hormone variant of claim 24 wherein said no acid residue is selected from the group consisting of Q46, N47, P48, Q49, T50, S51, K72, and V173, numbered from the N-terminus of 191-amino acid human growth hormone. 32. The human growth hormone variant of claim 31 wherein said amino acid substitution is selected from the group consisting of N47A and S51A. 33. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of R19, E30, E33, S43, F44, L73, F97, A98, N99, S100, L101, V102, Y103, G104, E186, S188, and F191, numbered from the N-terminus of 191-amino acid human growth hormone. 34. The human growth hormone variant of claim 33 wherein said amino acid residue is selected from the group consisting of F97, A98, N99, S100, L101, V102, Y103, and G104, numbered from the N-terminus of 191-amino acid human growth hormone. 35. The human growth hormone variant of claim 34 wherein said amino acid substitution is selected from the group consisting of F97G, F97E, F97M, F97A, F97R, F97Q, F97S, F97Y, F97W, F97L, F97I, F97V, A98G, A98E, A98M, A98F, A98R, A98Q, A98S, A98D, A98N, A98H, A98K, N99G, N99E, N99M, N99F, N99A, N99R, N99Q, N99S, N99D, N99K, N99Y, S100G, S100E, S100M, S100F, S100A, S100R, S100Q, S100H, S100D, S100N, S100K, S100Y, L101G, L101E, L101M, L101F, L101A, L101R, L101Q, L101S, L101I, L101V, L101Y, V102G, V102E, V102M, V102F, V102A, V102R, V102Q, V102S, V102I, V102T, V102L, V102Y, V102W, Y103G, Y103E, Y103M, Y103F, Y103A, Y103R, Y103Q, Y103S, Y103W, Y103L, Y103I, Y103V, G104E, G104M, G104F, G104A, G104R, G104Q, G104S, and G104P. 36. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of E33, Q49, T50, I58, R167, and R178, numbered from the N-terminus of 191-amino acid human growth hormone. 37. The human growth hormone variant of claim 36 said amino acid substitution is selected from the group consisting of R178K and R178N. 38. The human growth hormone variant of claim 24 wherein said amino acid residue is selected from the group consisting of F97, S100, L101, V102, Y103, and T175, numbered from the N-terminus of 191-amino acid human growth hormone. 39. The human growth hormone variant of claim 38 wherein said amino acid substitution is selected from the group consisting of F97A, S100A, L101A, V102A, Y103A, and T175S. 40. The human growth hormone variant of claim 24 further comprising an amino acid substitution at amino acid residue G120. 41. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of P2, N12, L15, R16, and R19. 42. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of T3, P5, S7, and L9. 43. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of E30, E33, S43, F44, and Q46. 44. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of N47, P48, Q49, T50, and F54. 45. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S55, E56, S57, I58, and P59. 46. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S62, N63, E66, Q68, and K70. 47. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of S71, L73, R77, L80, and F97. 48. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of A98, N99, S100, L101, and V102. 49. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of Y103, G104, D169, T175, and R178. 50. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino residue selected from the group consisting of Q181, C182, R183, S184, and V185. 51. The variant of claim 24, wherein the variant comprises an amino acid substitution at an amino acid residue selected from the group consisting of E186, G187, S188, and F191.