Compositions and methods for protein design

• US 20060160138A1
• Filed: 01/13/2006
• Published: 07/20/2006
• Est. Priority Date: 01/13/2005
• Status: Abandoned Application

• Current Assignee: Codon Devices Incorporated
• Original Assignee: Codon Devices Incorporated
• Inventors: Church, George, Baynes, Brian M.
• Application Number:
  US11/332,657
• Publication Number:
  US 20060160138A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/20 Protein or domain folding
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry
• CPC Class Codes: G16C 20/64 Screening of libraries

Abstract

In certain aspects the present invention provides methods and compositions related to rational protein design.

Claims

1. A method for producing a protein having a desired characteristic comprising vi) applying an algorithm to a protein scaffold to generate a plurality of possible variants;

vii) screening the plurality of variants in silico to produce a rank ordered list of variants;

viii) generating nucleic acid molecules having predefined sequences that encode at least 10 of the variants wherein the nucleic acid molecules are generated by a method comprising;

a) providing a pool of oligonucleotides comprising partially overlapping sequences that define the sequence of each of said nucleic acid molecules that encode said variants;

b) incubating said pool of oligonucleotides under hybridization conditions and at least one of the following conditions;

(i) ligation conditions, (2) chain extension conditions, or (iii) chain extension and ligation conditions, thereby forming nucleic acid constructs; and

c) separating constructs having said predefined sequences from constructs not having said predefined sequences, thereby forming the nucleic acid molecules that encode said variants; and

ix) causing expression of said nucleic acid molecules to produce said protein variants; and

x) screening the variants to identify variants having the desired characteristic.

2. The method of claim 1, wherein nucleic acids encoding at least 1000 of the variants are generated.

3. The method of claim 1, wherein nucleic acids encoding at least 10,000 of the variants are generated.

4. The method of claim 1, wherein the nucleic acids encoding the variants are at least 1000 bases in length.

5. The method of claim 1, wherein the nucleic acids encoding the variants are at least 5000 bases in length.

6. The method of claim 1, wherein the variants are produced in vitro.

7. The method of claim 1, wherein the nucleic acid molecules encoding the variants are prepared in a single pool.

8. The method of claim 1, wherein at least a portion of the sequence of one or more nucleic acids has been codon remapped to reduce the homology with at least one other nucleic acid.

9. The method of claim 1, wherein the oligonucleotides are synthesized on an array.

10. The method of claim 9, wherein the array comprises a solid support and a plurality of discrete features associated with said solid support, wherein each feature independently comprises a population of oligonucleotides collectively having a defined consensus sequence but in which no more than 10 percent of said oligonucleotides of said feature have the identical sequence.

11. The method of claim 1, wherein the method for generating the nucleic acid molecules further comprises an error reduction process.

12. The method of claim 1, wherein the nucleic acid molecules encoding the variants comprise sticky ends.

13. The method of claim 1, wherein one or more of the oligonucleotides that define the sequence of the nucleic acid molecules further comprise sequence tags such that a set of oligonucleotides that defines the sequence of a nucleic acid construct having a desired sequence has a distinguishable complement of sequence tags as compared to a set of oligonucleotides that defines the sequence of an incorrect product, and wherein nucleic acid constructs having a desired sequence are separated from incorrect crossover products based on size or electrophoretic mobility.

14. The method of claim 1, wherein a set of oligonucleotides that defines the sequence of a nucleic acid construct having a desired sequence forms sticky ends that permit circularization of the correctly formed product, and wherein correctly formed circularized products are separated from incorrectly formed linear products.

15. The method of claim 14, wherein the circularized products are separated from the linear products by digesting the linear products with an exonuclease.

16. The method of claim 1, wherein the nucleic acid molecules encoding the variants comprise vector sequences and sticky ends that permit circularization of the nucleic acid molecule to produce a circularized expression plasmid.

17. A biosynthetic library comprising a plurality of synthetic DNAs encoding a plurality of candidate proteins which can be selected or screened for species having a predetermined property or set of properties, the library comprising plural DNAs comprising regions of sequence homology and being assembled from chemically synthesized oligonucleotides.

18. A biosynthetic library comprising a plurality of synthetic DNAs encoding a plurality of candidate proteins which can be selected or screened for species having a predetermined property or set of properties, the library comprising plural DNAs chemically synthesized or assembled from chemically synthesized oligonucleotides and comprising reading frames of multiple said DNAs exploiting consistent codon usage patterns so as to promote similar expression levels in a selected expression system.

19. The library of claim 18, wherein said chemically synthesized oligonucleotides are synthesized in parallel.

20. The library of claim 18, wherein said DNAs are assembled in parallel from chemically synthesized oligonucleotides.