Capillary array-based enzyme screening

• US 6,972,183 B1
• Filed: 11/22/1999
• Issued: 12/06/2005
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: BASF Enzymes LLC (BASF SE)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Keller, Martin, Short, Jay M., Lafferty, William Michael
• Primary Examiner(s): Wessendorf, T. D.
• Application Number:
  US09/444,112
• Time in Patent Office: 2,206 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/7.4, 435/7.2, 435/4, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/7.4
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays

Abstract

A process for screening an expression library to identify clones expressing enzymes having a desired activity is provided. The process involves first generating from genomic DNA samples of one or more microorganisms an expression library comprising a plurality of recombinant cell clones, and then introducing into capillaries in a capillary array a substrate and at least a subset of the clones, either individually or as a mixture. Interaction of the substrate and a clone expressing an enzyme having the desired activity produces an optically detectable signal, which can then be spatially detected to identify capillaries containing clones producing such a signal. The signal-producing clones can then be recovered from the identified capillaries.

Claims

1. A method for identifying a clone expressing an enzyme, the method comprising:

(a) generating an expression library comprising a plurality of recombinant clones comprising nucleic acid sequences derived from genomic DNA samples of at least one microorganism;

(b) introducing a mixture of a substrate selected for being specific for an enzyme selected from lipases, esterases, proteases, peptidases, reductases, oxidoreductases, lyases, ligases, isomerases, polymerases, synthases, synthetases, glycosidases, transferases, phosphatases, kinases, mono-and dioxygenases, peroxidases, hydrolases, hydratases, nitrilases, transaminases, amidases and acylases and clones from the library into capillaries in a capillary array;

(c) incubating the clones with the substrate in the capillary-array in a reservoir containing water for a period of time to allow water to evaporate in the capillaries sufficient for at least one of the clones to express an enzyme that interacts with the substrate to produce an optically detectable signal;

(d) spatially detecting the signal to identify at least one capillary containing at least one signal-producing clone; and

(e) recovering the signal-producing clone from the identified capillary, thereby identifying a clone expressing an enzyme having the desired enzymatic activity.

2. The method of claim 1, wherein the expression library is generated from genomic DNA samples of at least one microorganism and the recombinant clones comprise host cells transformed with constructs comprising the nucleic acid sequences derived from the DNA samples.

3. The method of claim 2, wherein the microorganisms comprise prokaryotic cells.

4. The method of claim 2, wherein the microorganisms are a plurality of microorganisms.

5. The method of claim 2, wherein the microorganisms are derived from an environmental sample.

6. The method of claim 2, wherein the microorganisms are selected from the group consisting of terrestrial microorganisms, marine microorganisms and airborne microorganisms.

7. The method of claim 6, wherein the microorganisms comprise extremophiles.

8. The method of claim 7, wherein the extremophiles are thermophiles.

9. The method of claim 7, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles and acidophiles.

10. The method of claim 2, wherein the host cells are selected from the group consisting of bacterial cells, fungal cells, plant cells, insect cells and animal cells.

11. The method of claim 2, wherein the host cells are prokaryotic cells.

12. The method of claim 11, wherein the prokaryotic cells are bacterial cells.

13. The method of claim 12, wherein the bacterial cells are E, coli.

14. The method of claim 1, wherein the substrate is a chromogenic substrate.

15. The method of claim 1, wherein the substrate is a fluorogenic substrate.

16. The method of claim 15, wherein the signal is optical fluorescence.

17. The method of claim 15, wherein the fluorogenic substrate comprises umbelliferone.

18. The method of claim 15, wherein the fluorogenic substrate comprises resorufin.

19. The method of claim 15, wherein the fluorogenic substrate comprises fluorescein.

20. The method of claim 15, wherein the fluorogenic substrate comprises rhodamine.

21. The method of claim 1, wherein the detection is provided by a detector comprising a CCD, CID or photodiode array.

22. The method of claim 1, wherein the capillary array comprises at least about 100 capillaries.

23. The method of claim 1, wherein the capillary array comprises at least about 1000 capillaries.

24. The method of claim 1, wherein the capillary array comprises at least about 5000 capillaries.

25. The method of claim 1, wherein the substrate liquid and the clones are introduced simultaneously as a cell/substrate liquid mixture into capillaries in the capillary array.

26. The method of claim 1, further comprising biopanning prior to (b).

27. The method of claim 2, further comprising normalizing the genomic DNA prior to generating the expression library.

28. The method of claim 1, further comprising isolating at least one enzyme from the recovered clones.

29. A method for producing a recombinant enzyme having a desired enzyme activity, comprising:

(a) generating an expression library comprising a plurality of recombinant clones comprising nucleic acid sequences isolated from genomic DNA samples of at least one microorganism;

(b) introducing a mixture of a cell permeabilizing substrate specific for an enzyme selected from lipases, esterases, proteases, peptidases, reductases, oxidoreductases, lyases, ligases, isomerases, polymerases, synthases, synthetases, glycosidases, transferases, phosphatases, kinases, mono-and dioxygenases, peroxidases, hydrolases, hydratases, nitrilases, transaminases, amidases and acylases and recombinant clones from the library into capillaries in a capillary array;

(c) incubating the substrate and the clones in the capillaries to allow specific intracellular interaction of the substrate and a recombinant clone expressing the enzyme having the desired enzyme activity to produce an optically detectable signal;

(c) spatially detecting the signal to identify at least one capillary containing at least one signal-producing recombinant clone;

(d) recovering the signal-producing recombinant clone from identified capillaries;

(e) isolating a nucleic acid sequence from a signal-producing recombinant clone, wherein the nucleic acid sequence encodes an enzyme having the desired specific enzyme activity;

(f) inserting the nucleic acid sequence isolated in (e) into a suitable expression vector to produce a transformable construct;

(g) transforming a suitable host cell with a sequence comprising the transformable construct produced in (f) to produce a recombinant cell; and

(h) recovering from the recombinant cell produced in (g) a recombinant enzyme having the desired enzyme activity expressed by the recombinant cell.