Compositions and methods for protein production

Patent metadata

Field	Value
Publication number	US-9605258-B2
Application number	US-201514593707-A
Country	US
Kind code	B2
Filing date	Jan 9, 2015
Priority date	Jun 11, 2010
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

Abstract

The present invention provides a method of selecting a mRNA for production of a polypeptide of interest comprising: a) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest; b) determining one or more or two or more of the following parameters for each individual mRNA sequence of (a): (i) minimum free energy (MFE) RNA secondary structure; (ii) ensemble free energy (EFE); (iii) frequency of the minimum free energy (FMFE) RNA secondary structure in a thermodynamic ensemble; and (iv) ensemble diversity (ED); c) ranking the individual mRNA sequences of the array according the parameters determined in step (b); and d) selecting a mRNA sequence from the ranked array of step (c), wherein the selected mRNA produces the polypeptide of interest. The present invention further provides a method of selecting a mRNA for enhanced and reduced production of a polypeptide of interest.

First claim

That which is claimed is:

1. A method for modifying the production of a polypeptide of interest in a cell of an organism, the method comprising:
(a) selecting an mRNA encoding a polypeptide of interest, comprising:
i) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest, wherein the individual mRNA sequences comprise nucleotides from positions −20 through 50 relative to the start codon;
ii) determining minimum free energy (MFE) RNA secondary structure for each individual mRNA sequence of (i);
iii) ranking the individual mRNA sequences of the array according to the MFE RNA secondary structure determined in (ii) from highest MFE RNA secondary structure to lowest MFE RNA secondary structure; and
iv) selecting a mRNA sequence from the ranked array of step (iii), wherein the selected mRNA produces the polypeptide of interest in a eukaryotic organism, and at least steps i) to iii);

(b) mutating the nucleotide sequence of the mRNA of (a) to produce a mutated mRNA having an enhanced or reduced MFE RNA secondary structure as compared to the MFE RNA secondary structure of the nucleotide sequence of the mRNA of (a));
wherein the mutated mRNA encodes a mutated polypeptide sequence as compared to the polypeptide sequence encoded by the mRNA of (a), the mutated polypeptide sequence retains the function of the polypeptide encoded by the mRNA of (a), and expression of the mutated mRNA sequence results in modified production of the polypeptide of interest as compared to the wild-type mRNA sequence.

2. The method of claim 1 , wherein the individual mRNA sequences comprise nucleotides at positions −15 through 40 relative to the start codon.

3. The method of claim 1 , wherein the individual mRNA sequences comprise nucleotides at positions −4 through 35 relative to the start codon.

4. The method of claim 1 , wherein the mRNA is selected for enhanced production of a polypeptide of interest.

5. The method of claim 4 , wherein the mRNA is selected from the group of mRNAs having an MFE in the range from about 0 kcal/mol to about −2 kcal/mol, wherein expression of the selected mRNA sequence results in enhanced production of the polypeptide of interest as compared to the wild-type mRNA sequence.

6. The method of claim 1 , wherein the mRNA is selected for reduced production of a polypeptide of interest.

7. The method of claim 6 , wherein the mRNA is selected from the group of mRNAs having an MFE in the range from about −9 kcal/mol to about −18 kcal/mol, wherein expression of the selected mRNA sequence results in reduced production of the polypeptide of interest as compared to the wild-type mRNA sequence.

8. The method of claim 1 , wherein the polypeptide of interest comprises a leader peptide.

9. A method for modifying the production of a polypeptide of interest in a cell of an organism, the method comprising:
(a) selecting an mRNA encoding a polypeptide of interest, comprising
i) producing an array of individual mRNA sequences comprising different nucleotide sequences encoding the polypeptide of interest, wherein the individual mRNA sequences comprise nucleotides from positions −20 through 50 relative to the start codon;
ii) determining minimum free energy (MFE) RNA secondary structure for each individual mRNA sequence of (i);
iii) ranking the individual mRNA sequences of the array according to the MFE RNA secondary structure determined in (ii) from highest MFE RNA secondary structure to lowest MFE RNA secondary structure; and
iv) selecting a mRNA sequence from the ranked array of step (iii),

wherein the selected mRNA produces the polypeptide of interest in a eukaryotic organism, and at least steps i) to iii);
(b) identifying the differences between the nucleotide sequence of the mRNA selected according to (a) and a wild-type endogenous nucleotide sequence of said organism, wherein the wild-type endogenous nucleotide sequence encodes a polypeptide of interest that is the same as that encoded by the mRNA selected according to (a); and
(c) mutating in situ the endogenous nucleotide sequence encoding the polypeptide of interest to incorporate each of the nucleotide differences identified in (b) into the endogenous nucleotide sequence, whereby the production of the polypeptide of interest from the in situ mutated endogenous nucleotide sequence in the cell of the organism is modified.

10. The method of claim 9 , wherein the individual mRNA sequences comprise nucleotides at positions −15 through 40 relative to the start codon.

11. The method of claim 9 , wherein the individual mRNA sequences comprise nucleotides at positions −4 through 35 relative to the start codon.

12. The method of claim 9 , wherein the mRNA is selected for enhanced production of a polypeptide of interest.

13. The method of claim 12 , wherein the mRNA is selected from the group of mRNAs having an MFE in the range from about 0 kcal/mol to about −2 kcal/mol, wherein expression of the selected mRNA sequence results in enhanced production of the polypeptide of interest as compared to the wild-type mRNA sequence.

14. The method of claim 9 , wherein the mRNA is selected for reduced production of a polypeptide of interest.

15. The method of claim 14 , wherein the mRNA is selected from the group of mRNAs having an MFE in the range from about —9 kcal/mol to about —18 kcal/mol, wherein expression of the selected mRNA sequence results in reduced production of the polypeptide of interest as compared to the wild-type mRNA sequence.

16. The method of claim 9 , wherein the polypeptide of interest comprises a leader peptide.

Assignees

• Syngenta Participations Ag

Inventors

• Basu Shib Sankar
• Dang Kristen Kamerath
• Azhakanandam Kasimalai
• Mcneill Thomas Z
• Guo Sheng

Classifications

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title

• C12N15/1089 Primary

  Design, preparation, screening or analysis of libraries using computer algorithms · CPC title