METHODS FOR SCREENING PROTEINS USING DNA ENCODED CHEMICAL LIBRARIES AS TEMPLATES FOR ENZYME CATALYSIS

• US 20130252849A1
• Filed: 03/13/2013
• Published: 09/26/2013
• Est. Priority Date: 03/21/2012
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Hudson, Michael E.
• Granted Patent: US 9,150,853 B2
• Time in Patent Office: Days
• US Class Current: 506/11
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• 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

Abstract

Disclosed are methods, compositions and devices for screening a protein library for proteins having a desired activity, such as capable of catalyzing the formation of a bond between two reactants. In an exemplary embodiments, a plurality of proteins are expressed in vitro from a plurality of nucleic acids, the plurality of proteins are exposed with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore, the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes is detected and the complexes showing detectable fluorescence energy transfer are isolated, thereby isolating proteins having the desired enzymatic activity.

Claims

1. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) providing a plurality of nucleic acids, each nucleic acid having a predefined sequence, at a distinct feature of a solid support;

b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;

c) exposing each of the plurality of proteins with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore;

d) detecting the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes; and

e) isolating the complexes showing detectable fluorescence energy transfer, thereby isolating the proteins having the desired enzymatic activity.

2. The method of claim 1 wherein the desired activity comprises formation of a bond between the two reactants.

3. The method of claim 1 wherein the protein having the desired activity specifically binds the two reactants.

4. The method of claim 1 wherein the reactant is at one end of the oligonucleotide and the fluorophore is at another end of the oligonucleotide.

5. The method of claim 1 wherein each feature is a well of a microwell plate.

6. A method for screening a protein library for a protein having desired activity, the method comprising:

a) providing a plurality of nucleic acids, each nucleic acid having a predefined sequence, at a distinct feature of a solid support;

b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;

c) exposing each of the plurality of proteins with two oligonucleotides, each oligonucleotide having a reactant at one end, wherein proteins having the desired activity bind the two reactants thereby forming a complex with the two oligonucleotides; and

d) providing a ligase, wherein the ligase catalyses the ligation of free ends of the two oligonucleotides complexed with the proteins having the desired activity.

7. The method of claim 6 wherein the two oligonucleotides are double stranded oligonucleotides.

8. The method of claim 6 wherein the two oligonucleotides are single stranded oligonucleotides.

9. The method of claim 7 further comprising adding a helper oligonucleotide to capable of bridging the two single stranded oligonucleotides bound to the protein having the desired activity.

10. The method of claim 6 wherein each feature is a well of a microwell plate.

11. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) incubating a host cell comprising a first nucleic acid sequence encoding a plurality of proteins fused to aga-2 and a second nucleic acid sequence encoding a plurality of transcription activator like-effector fused with aga-1, under conditions sufficient for expressing the protein and the transcription activator like-effector as a protein complex at the cell surface;

b) exposing the protein complex to a first single stranded oligonucleotide having a first reactant, wherein the oligonucleotide binds to the transcription activator like-effector;

c) exposing the protein complex bound to the transcription activator like-effector, to a second single stranded oligonucleotide having a second reactant and a detectable label, wherein the second single stranded oligonucleotide has a sequence complementary to the first single stranded oligonucleotide; and

d) detecting the label at the surface of the host cell, wherein the detection of the label is correlated to a presence of the protein having the desired activity.

12. The method of claim 11 wherein the host cell is a yeast cell.

13. The method of claim 11 further comprising isolating host cells expressing proteins having the desired activity.

14. A method for screening a protein library for a protein having a desired activity, the method comprising:

a) providing a solid surface having a plurality of anchor oligonucleotides capable of hybridizing with a plurality of double stranded nucleic acids encoding a plurality of proteins;

b) hybridizing a single stranded oligonucleotide having a first reactant to a free end of the double stranded nucleic acids;

c) expressing in vitro a plurality of proteins from the plurality of double stranded nucleic acids in presence of a reactant having a detectable label; and

d) identifying features having the detectable label, wherein the detection of the label is correlated to a presence of the proteins having the desired activity.

15. The method of claim 14 wherein the plurality of double stranded oligonucleotides encodes for a library of proteins.

16. The method of claim 15 wherein the library of proteins is a library of protein variants.

17. The method of claim 14 wherein the step of expressing is in presence of a library of reactants.

18. The method of claim 14 wherein the protein having a desired activity catalyze a condensation reaction.