Crystallization of IGF-1

• US 7,084,240 B2
• Filed: 02/01/2002
• Issued: 08/01/2006
• Est. Priority Date: 02/09/2001
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ultsch, Mark, Schaffer, Michelle, Vajdos, Felix
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US10/066,009
• Publication Number:
  US 20020165155A1
• Time in Patent Office: 1,642 Days
• Field of Search: 530/300, 530/344, 436/86, 702/19, 702/27
• US Class Current: 530/300
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/65 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Abstract

Crystalline IGF-1 is provided along with a method for production thereof. Crystallizing IGF-1 comprises the steps of mixing an aqueous solution comprising IGF-1 with a reservoir solution comprising a precipitant to form a mixture; and crystallizing the mixture, optionally also recrystallizing and isolating the crystalline IGF-1. In addition, a method for identifying IGF-1 indirect agonists is provided using a detergent as a standard for the level of inhibition of binding of IGFBP-1 or IGFBP-3 to IGF-1 and/or using the coordinates of the binding pockets of IGF-1 to which a candidate indirect agonist binds for structure-based drug design.

Claims

1. A crystal formed by insulin-like growth factor-1 (IGF-1) of SEQ ID NO:

1 that diffracts x-ray radiation to produce a diffraction pattern representing the three-dimensional structure of the IGF-1, and has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

.

2. The crystal of claim 1 wherein the IGF-1 contains an A-, B-, C-, and D-region and forms a dimer in the crystal and wherein the crystal comprises a receptor binding site at the dimer interface.

3. A composition comprising the crystal of claim 1 and a carrier.

4. The composition of claim 3 wherein the IGF-1 is biologically active when resolubilized.

5. A heavy-atom derivative of of the crystalline IGF-1 of claim 1.

6. A co-crystalline complex of crystals of insulin-like growth factor-1 (IGF-1) of SEQ ID NO:

1 having approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

, and N,N-bis(3-D-gluconamidopropyl)-deoxycholamine.

7. A method of crystallizing insulin-like growth factor-1 (IGF-1) of SEQ ID NO:

1 comprising the steps of;

(a) mixing (1) an aqueous solution comprising said IGF-1 in about 0.15 M sodium chloride and about 20 mM sodium acetate (pH 4.5), up to a final IGF-1 concentration of about 5to 15 mg/ml, with (2) reservoir solution consisting of about 24% polyethylene glycol 3350 buffered to about pH 6.5 with about 0.1 M sodium cacodylate and about 14mM N,N-bis(3-D-gluconamidopropyl)-deoxycholamine, as a detergent, in a ratio of about 4;

5, to form a mixed volume;

(b) allowing said mixed volume to equilibrate over said reservoir solution until small crystals with a plate-like morphology appear in about 4 to 5 days;

(c) adding methyl pentanediol to a final concentration of about 20%; and

(d) recrystallizing the mixture to yield IGF-1 crystals that diffract x-ray radiation to produce a diffraction pattern representing the three-dimensional structure of the IGF-1, and have approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

.

8. The method of claim 7 wherein the IGF-1 is obtained from a prokaryotic cell.

9. The method of claim 7 wherein the equilibration in step (b) is carried out by vapor diffusion crystallization, batch crystallization, liquid bridge crystallization or dialysis crystallization.

10. The method of claim 7 wherein the equilibration in step (b) is carried out by vapor diffusion crystallization.

11. Crystalline insulin-like growth factor-1 (IGF-1) produced by the method of claim 7.

12. A method for determining a three-dimensional structure of IGF-1 of SEQ ID NO:

1 comprising;

(a) crystallizing the IGF-1;

(b) irradiating the crystalline IGF-1 to obtain a diffraction pattern characteristic of the crystalline IGF-1, wherein the crystalline IGF-1 has approximately the following cell constants a=31.831 Å

, b=71.055 Å

, c=65.995 Å

, and a space group of C222₁, and α

=β

=γ

; and

(c) transforming the diffraction pattern into the three-dimensional structure of the IGF-1.