Isolation of enzymes

• US 6,184,012 B1
• Filed: 04/22/1999
• Issued: 02/06/2001
• Est. Priority Date: 04/25/1996
• Status: Expired due to Fees

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Neri, Dario, Viti, Francesca, Huber, Adrian, Winter, Gregory Paul, Demartis, Salvatore, Tawfik, Dan S
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US09/142,355
• Time in Patent Office: 656 Days
• Field of Search: 435/183, 435/188
• US Class Current: 435/188
• CPC Class Codes: C07K 14/4728 Calcium binding proteins, e.g. calmodulin
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/0002 Antibodies with enzymatic activity, e.g. abzymes
• CPC Class Codes: C12N 9/1088 Glutathione transferase (2.5.1.18)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

The present invention relates to a method of isolating an enzyme having a desired chemical or biological activity, the method comprising linking the enzyme to a substrate for the enzyme, reacting the enzyme and substrate such that a product is produced which remains linked to the enzyme and isolating the enzyme by selectively isolating the product linked to the enzyme.

Claims

1. A method of isolating an enzyme having a desired chemical or biological activity the method comprising:

a) linking the enzyme to a substrate for the enzyme

b) reacting the enzyme and substrate such that a product is produced which remains linked to the enzyme; and

c) isolating the enzyme by selectively isolating the product linked to the enzyme.

2. A method according to claim 1 in which the enzyme and substrate are linked via a binding pair.

3. A method according to claim 2 in which the binding pair are a calcium dependent binding polypeptide and a ligand thereof.

4. A method according to claim 3 in which the calcium dependent binding polypeptide is calmodulin or a derivative of calmodulin that retains binding activity, and the ligand of the calcium dependent polypeptide is a calmodulin ligand or derivative of a calmodulin ligand that retains calmodulin binding activity.

5. A method according to claim 2 in which the binding pair bind with a dissociation constant (K_d) of 10 nM or less, measured at a pH of between 6 and 9 at 20°

C.

6. A method according to claim 5 wherein the dissociation constant is 1 nM or less.

7. A method according to claim 1 in which the enzyme and substrate are linked covalently.

8. A method according to claim 1 wherein the enzyme is displayed on a support containing a DNA encoding the enzyme.

9. A method according to claim 8, wherein the support is a bacteriophage particle.

10. A method according to claim 1 wherein the enzyme is isolated by binding the product to a solid matrix.

11. A method according to claim 10, wherein the solid matrix is suitable for biopanning.

12. A method according to claim 10 wherein the solid matrix is suitable for affinity chromatography.

13. A method according to claim 10, wherein the product is bound to the solid matrix by an antibody bound to the solid matrix.

14. A method according to claim 10 comprising a further step of dissociating the enzyme from the product bound to the solid matrix.

15. A method according to claim 10 wherein the dissociation of enzyme from the product is achieved by the use of a calcium chelator.

16. A method according to claim 1 wherein the enzyme is produced in an expression library.

17. A method according to claim 1 wherein the enzyme is produced by mutagenesis of a nucleic acid sequence.