Rational directed protein evolution using two-dimensional rational mutagenesis scanning

• US 20060020396A1
• Filed: 08/02/2005
• Published: 01/26/2006
• Est. Priority Date: 09/09/2002
• Status: Abandoned Application

• Current Assignee: HanAll Biopharma Co. Ltd.
• Original Assignee: HanAll Biopharma Co. Ltd.
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene, Ramos, Hugo Cruz
• Application Number:
  US11/196,067
• Publication Number:
  US 20060020396A1
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• 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 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis

Abstract

Rational methods for generating protein variants are provided. Processes and systems for the high throughput directed evolution of peptides and proteins employing the method are provided.

Claims

1. A method for increasing serum stability of therapeutic target proteins, comprising modifying a target protein to exhibit increased resistance to protease digestion, thereby increasing serum stability of the target protein.

2. The method of claim 1, wherein modification of the protein is effected by:

selecting, on the target protein, target amino acids susceptible to proteolysis;

replacing each target amino acid with a replacement amino acid amenable to decreased proteolysis to form a candidate lead protein, wherein one amino acid replacement occurs on each target protein;

expressing each candidate lead protein in a cell contained in an addressable array; and

assaying each candidate lead protein for the presence of increased resistance to proteolysis, thereby identifying proteins that are Leads, wherein Leads exhibiting increased resistance to proteolysis have increased serum half-life compared to the wild-type target protein.

3. The method of claim 2, wherein the target protein is a cytokine.

4. The method of claim 3, wherein the cytokine is IFNα

-2b and IFN-β

.

5. The method of claim 2, wherein:

the selection of the one or more target amino acids is conducted in silico and the target amino acids are designated is-HITS.

6. The method of claim 5, wherein:

in silico selection further comprises selecting an is-HIT target residue that is susceptible to digestion by one or more proteases.

7. The method of claim 6, wherein the Leads possess increased resistance to proteolysis compared to the wild-type target protein.

8. The method of claim 2, wherein the method is repeated and more than one amino acid replacement occurs on each target protein.

9. The method of claim 1, wherein modification of the protein is effected by:

identifying, within a target protein or peptide, target amino acids amenable to providing resistance to proteolysis upon amino acid replacement, wherein each target amino acid is designated as an in silico-HIT (is-HIT);

identifying one or more replacement amino acids, specific for each is-HIT, amenable to providing the resistance to proteolysis to the target upon amino acid replacement of the target protein or peptide, wherein each single amino acid replacement within the target protein or peptide is designated as a candidate lead protein;

producing a population of sets of nucleic acid molecules that encode each of the candidate lead proteins, wherein each candidate lead protein comprises a single amino acid replacement, and wherein each polynucleotide in a set encodes a candidate lead protein that differs by one amino acid from the target protein or peptide;

introducing each set of nucleic acid molecules into host cells and expressing the encoded candidate lead proteins, wherein the host cells are present in an addressable array; and

individually screening the sets of encoded candidate lead proteins to identify one or more proteins that has activity that differs from the target protein, wherein each such protein is designated a Lead mutant protein.

10. The method of claim 9, wherein the target protein is a cytokine.

11. The method of claim 10, wherein the cytokine is IFNα

-2b and IFN-β

.

12. The method of claim 9, wherein:

the nucleic acid molecules are produced by site-specific mutagenesis.

13. The method of claim 9, further comprising:

recombining the nucleic acid molecules that encode the Leads, to produce a set of candidate SuperLeads comprising a combination of two or more single amino acid mutations derived from two or more LEAD mutant proteins;

introducing the each set of candidate SuperLeads into cells; and

individually screening the sets of cells that encode the candidate SuperLeads to identify one or more cells that encodes a protein that has activity that differs from the target protein and has properties that differ from the original Leads, wherein each such protein is designated a SuperLead.

14. The method of claim 13, wherein:

the nucleic acid molecules are produced by a method selected from among Additive Directional Mutagenesis, multi-overlapped primer extensions, oligonucleotide-mediated mutagenesis, nucleic acid shuffling, recombination, site-specific mutagenesis and de novo synthesis.

15. The method of claim 13, wherein the recombining is two, three or more up to all of the nucleic acids encoding the Leads.

16. The method of claim 9, wherein each is-HIT target amino acid is susceptible to digestion by one or more proteases.

17. The method of claim 16, wherein the Leads or SuperLeads possess increased resistance to proteolysis compared to the wild-type target protein.

18. The method of claim 9, wherein the method is repeated and more than one amino acid replacement occurs on each target protein.