High throughput screening for novel enzymes

• US 20010034031A1
• Filed: 05/03/2001
• Published: 10/25/2001
• Est. Priority Date: 06/16/1997
• Status: Active Grant

• Current Assignee: BASF Enzymes LLC (BASF SE)
• Original Assignee: Recombinant Biocatalysis Incorporated Delaware Corporation
• Inventors: Keller, Martin, Short, Jay M.
• Granted Patent: US 6,806,048 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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

Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring co-encapsulation, a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.

Claims

1. A method of screening for an agent that modulates the interaction of a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, comprising co-encapsulating the agent with the first test protein and second test protein in a suitable microenvironment and determining the ability of the agent to modulate the interaction of the first test protein linked to a DNA binding moiety with the second test protein covalently linked to a transcriptional activation moiety, wherein the agent enhances or inhibits the expression of a detectable protein, and wherein the enhancement or inhibition is detected by FACS analysis.

2. The method of

claim 1

, wherein the agent is an enzyme or small molecule.

3. The method of

claim 2

, wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.

4. The method of

claim 1

, wherein the agent inhibits the activity of the first protein or the second protein.

5. The method of

claim 1

, wherein the agent enhances the activity of the first protein or the second protein.

6. The method of

claim 1

, wherein the agent is expressed from a recombinant cell co-encapsulated with the recombinant cell expressing the target protein and detectable marker.

7. The method of

claim 6

, wherein the recombinant cell is a eukaryotic cell.

8. The method of

claim 6

, wherein the recombinant cell is a prokaryotic cell.

9. The method of

claim 1

, wherein the micro-environment is a liposome, gel microdrop, bead, agarose, cell, ghost red blood cell or ghost macrophage.

10. The method of

claim 9

, wherein the liposomes are prepared from one or more phospholipids, glycolipids, steroids, alkyl phosphates or fatty acid esters.

11. The method of

claim 10

, wherein the phospholipids are selected from the group consisting of lecithin, sphingomyelin and dipalmitoyl.

12. The method of

claim 10

, wherein the steroids are selected from the group consisting of cholesterol, chlorestanol and lanosterol.

13. The method of

claim 1

, wherein the detectable marker is a fluorescent dye, a visible dye, a bioluminescent material, a chemiluminescent material, a radioactive material, or an enzymatic substrate.

14. The method of

claim 13

, wherein the bioluminescent material is green fluorescent protein (GFP) or red fluorescent protein (RFP).

15. The method of

claim 13

, wherein detection of the fluorescent dye or a visible dye is carried out by fluorometric or spectrophotometric measurement.