Compositions for adjustable ribosome translation speed and methods of use

Patent metadata

Field	Value
Publication number	US-11293028-B2
Application number	US-201615765132-A
Country	US
Kind code	B2
Filing date	Oct 3, 2016
Priority date	Oct 2, 2015
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

Abstract

The present invention relates to the effects of codon context and synonymous codon changes on mRNA translation and methods of increasing protein production.

First claim

What is claimed is:

1. A recombinant nucleic acid molecule comprising a mutant triacontanucleotide sequence having at least ten codons, wherein at least one of the sixth, seventh, eighth, ninth and tenth codons, in the 5′ to 3′ direction, are synonymous codons, and wherein the mutant triacontanucleotide sequence is selected from SEQ ID NOs: 8-15.

2. The recombinant molecule of claim 1 , wherein the mutant triacontanucleotide sequence is operably linked to a heterologous polynucleotide sequence that encodes a polypeptide of interest.

3. The recombinant molecule of claim 2 , wherein the polypeptide of interest is insulin.

4. A vector comprising the recombinant molecule of claim 1 .

5. An isolated host cell comprising the recombinant molecule of claim 1 .

6. A vector comprising the recombinant molecule of claim 2 .

7. An isolated host cell comprising the recombinant molecule of claim 2 .

8. A method of modulating protein production within a host cell, comprising culturing a host cell under conditions sufficient for protein expression, wherein the recombinant molecule of claim 4 has been stably introduced into the host cell.

9. A method of increasing the translation speed of a polynucleotide sequence, comprising providing to a host cell a polynucleotide sequence that encodes a protein and is operably linked to a wild type triacontanucleotide sequence comprising ten codons and encoding SEQ ID NO:1, and mutating the triacontanucleotide sequence so that the mutant triacontanucleotide sequence is selected from SEQ ID NOs: 8-15, and culturing the host cell under conditions to express the polynucleotide sequence.

10. A method for increasing specific cellular productivity of a polypeptide of interest in a host cell comprising: (a) providing a polynucleotide sequence that encodes the polypeptide of interest and is operably linked to a wild type triacontanucleotide sequence comprising ten codons and encoding SEQ ID NO:1, (b) mutating the triacontanucleotide sequence so that the mutant triacontanucleotide sequence is selected from SEQ ID NOs: 8-15; (c) introducing the polynucleotide sequence into the host cell; (d) incubating the cell under conditions wherein the introduced polynucleotide is expressed; and (e) isolating the polypeptide of interest.

Assignees

• Univ Utah Res Found

Inventors

• Hughes Kelly T
• Chevance Fabienne

Classifications

• C07K14/245

  Escherichia (G) · CPC title

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K14/62

  Insulins · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title