Protein Arrays and Methods of Using and Making the Same

• US 20130296192A1
• Filed: 11/10/2011
• Published: 11/07/2013
• Est. Priority Date: 11/12/2010
• 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., Chu, Larry Li-Yang
• Granted Patent: US 10,457,935 B2
• Time in Patent Office: Days
• US Class Current: 506/11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00509 Microcolumns
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

Methods and devices are provided for preparing a protein array having a plurality of proteins. In one embodiment, the method includes providing a plurality of nucleic acids each having a predefined sequence and expressing in vitro a plurality of proteins from the plurality of nucleic acids. In another embodiment, protein arrays having a solid surface and a microvolume are also provided. The solid surface can have a plurality of anchor oligonucleotides capable of hybridizing with a plurality of nucleic acids. The microvolume can cover each of the plurality of anchor oligonucleotides and can be configured to produce a polypeptide from each of the plurality of nucleic acids.

Claims

1. A method for preparing a protein array having a plurality of proteins, the method comprising:

(a) providing a plurality of nucleic acids each having a predefined sequence; and

(b) expressing in vitro a plurality of proteins from the plurality of nucleic acids, wherein the plurality of proteins are expressed on an array.

2. The method of claim 1 further comprising (c) measuring an activity of each of the plurality of proteins.

3. The method of claim 1, wherein the plurality of nucleic acids are synthesized on a solid surface.

4. The method of claim 1, wherein each of the plurality of nucleic acids comprises a regulatory genetic sequence.

5. The method of claim 1, wherein each of the plurality of proteins is expressed in vitro in a well of a micro-well plate.

6. The method of claim 1, wherein each of the plurality of proteins is expressed in vitro at a different feature of a solid surface.

7. A method for preparing a protein array having a plurality of proteins, the method comprising:

(a) providing a first microvolume comprising a population of nucleic acids having a plurality of distinct, predefined sequences;

(b) immobilizing the nucleic acid sequences onto an array comprising a plurality of anchor oligonucleotides having a sequence complementary to a terminus sequence of the nucleic acids;

(c) expressing in vitro in a second microvolume a plurality of proteins from the population of nucleic acids.

8. A method for producing at least one protein, the method comprising:

(a) providing a support having a plurality of distinct features, each feature comprising a plurality of immobilized anchor oligonucleotides;

(b) generating at least one plurality of nucleic acid having a predefined sequence onto the plurality of anchor oligonucleotides;

(c) providing a microvolume onto at least one feature of the support; and

(d) expressing in vitro in the microvolume the at least one protein from the at least one nucleic acid.

9. The method of claim 8 wherein each feature of the support comprises a distinct plurality of support-bound anchor oligonucleotides, wherein the 5′

end of each of the plurality of anchor oligonucleotide is complementary to the 5′

end of a distinct nucleic acid having a predefined sequence.

10. The method of claim 8 wherein the plurality of nucleic acids are generated by assembling a plurality of construction oligonucleotides comprising partially overlapping sequences that define the sequence of the at least one nucleic acid.

11. The method of claim 10 wherein the at least one nucleic acid is generated under (i) ligation conditions, (ii) chain extension conditions, or (iii) chain extension and ligation conditions.

12. The method of claim 8 wherein the microvolume comprises reagents appropriate for expressing in vitro the at least one protein from the at least one nucleic acid.

13. The method of claim 8 further comprising verifying the at least one nucleic acid sequence prior to the step of expressing the at least one protein.

14. The method of claim 8 further comprising:

synthesizing a plurality of partially overlapping construction oligonucleotides, wherein each construction oligonucleotide is synthesized at a distinct feature of the support comprising immobilized complementary construction oligonucleotides;

releasing the construction oligonucleotides in at least one microvolume; and

transferring the at least one microvolume to a distinct feature comprising a plurality of anchor oligonucleotides.

15. The method of claim 8 wherein at least 1,000 proteins are produced.

16. The method of claim 8 wherein the proteins are proteins variants.

17. The method of claim 8 further comprising screening the at least one protein to identify proteins having a desired characteristic.

18. A protein array comprising:

(a) a solid surface having a plurality of anchor oligonucleotides capable of hybridizing with a plurality of nucleic acids; and

(b) a microvolume covering each of the plurality of anchor oligonucleotides, the microvolume configured to produce a polypeptide from each of the plurality of nucleic acids.

19. A protein array comprising:

(a) a first plurality of features on a support, each of the first plurality of features comprising a plurality of immobilized single stranded oligonucleotides, wherein the plurality of single stranded oligonucleotides comprises partially overlapping sequences that define the sequence of each of a plurality of nucleic acid molecules encoding a plurality of proteins; and

(b) a second plurality of features, the second plurality of features comprising a plurality of anchor oligonucleotides having a sequence complementary to a terminus sequence of each of the plurality nucleic acids.