Protein engineering

9,927,440

12/954317

November 24, 2010

Methods of optimizing mRNA sequences for expression in host cells are provided. Methods of determining the stability of a protein are also provided. Methods of determining the affinity of a ligand for a protein are also provided.

Hellinga, Homme W. (Durham, NC, US); Allert, Malin J. (Raleigh, NC, US)

Duke University (Durham, NC, US)

1. A method of producing an optimized mRNA sequence for protein expression in prokaryotes, comprising: (i) selecting an mRNA sequence comprising an AU composition of less than 57% in the last 35 nucleotides of a protein coding region thereof; (ii) altering the AU composition of the last 35 nucleotides of the protein coding region of the selected mRNA sequence to be at least 57% without changing the encoded amino acid sequence to produce an optimized mRNA sequence; and (iii) constructing an mRNA molecule comprising the optimized mRNA sequence.

2. A method of producing an optimized mRNA sequence for protein expression in prokaryotes, comprising: (i) selecting an mRNA sequence comprising an AU composition of less than 57% in the last 35 nucleotides of a protein coding region thereof; (ii) altering the AU composition of the last 35 nucleotides of the protein coding region of the selected mRNA sequence to be at least 57% without changing the encoded amino acid sequence, determining whether the AU composition of the first 35 nucleotides of a protein coding region of the mRNA sequence is less than 55%; altering the AU composition of the first 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence if the AU composition of the first 35 nucleotides of the coding region is less than 55%, such that the AU composition of the first 35 nucleotides of the coding region of the mRNA sequence is at least 55%; and altering the sequence of a first 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence, such that the first 35 nucleotides of the coding region if the mRNA sequence is predicted to have less secondary structure relative to the first 35 nucleotides of the coding region of the selected mRNA sequence, wherein the coding region further comprises nucleotides between the first 35 nucleotides of the coding region and the last 35 nucleotides of the coding region, to produce an optimized mRNA sequence; and (iii) constructing an mRNA molecule comprising the optimized mRNA sequence.

3. A method of producing an optimized mRNA sequence for protein expression in prokaryotes, comprising: (i) selecting an mRNA sequence comprising an AU composition of less than 57% in the last 35 nucleotides of a protein coding region thereof; (ii) altering the AU composition of the last 35 nucleotides of the protein coding region of the selected mRNA sequence to be at least 57% without changing the encoded amino acid sequence, and at least two of determining whether the AU composition of the first 35 nucleotides of the protein coding region of the mRNA sequence is less than 55%; altering the AU composition of the first 35nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence if the composition of the first 35 nucleotides of the coding region is less than 55%, such that the AU composition of the first 35 nucleotides of the coding region of the mRNA sequence is at least 55%; altering the sequence of a first 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence, such that the first 35 nucleotides of the coding region of the mRNA sequence is predicted to have less secondary structure relative to the first 35 nucleotides of the coding region of the selected mRNA sequence; altering the sequence of a last 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence, such that the last 35 nucleotides of the coding region of the mRNA sequence is predicted to have less secondary structure relative to the last 35 nucleotides of the coding region of the selected mRNA sequence; altering the sequence of the coding region of the selected mRNA sequence such that at least one codon is replaced with a codon that is used at higher frequency in a selected host cell; or altering the AU composition of a middle portion of the selected mRNA sequence, wherein the middle portion is between the first 35 nucleotides and the last 35 nucleotides of the coding region, to produce an optimized mRNA sequence; and (iii) constructing an mRNA molecule comprising the optimized mRNA sequence.

4. A method of producing an optimized mRNA sequence for protein expression in prokaryotes, comprising: (i) selecting an mRNA sequence comprising an AU composition of less than 57% in the last 35nucleotides of a protein coding region thereof; (ii) altering the AU composition of the last 35 nucleotides of the protein coding region of the selected mRNA sequence to be at least 57% without changing the encoded amino acid sequence, and at least three of: determining whether the AU composition of the first 35 nucleotides of the protein coding region of the mRNA sequence is less than 55%; altering the AU composition of the first 35nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence if the composition of the first 35 nucleotides of the coding region is less than 55%, such that the AU composition of the first 35 nucleotides of the coding region of the mRNA sequence is at least 55%; altering the sequence of a first 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence, such that the first 35 nucleotides of the coding region of the mRNA sequence is predicted to have less secondary structure relative to the first 35 nucleotides of the coding region of the selected mRNA sequence; altering the sequence of a last 35 nucleotides of the coding region of the selected mRNA sequence without changing the encoded amino acid sequence, such that the last 35 nucleotides of the coding region of the mRNA sequence is predicted to have less secondary structure relative to the last 35 nucleotides of the coding region of the selected mRNA sequence; altering the sequence of the coding region of the selected mRNA sequence such that at least one codon is replaced with a codon that is used at higher frequency in a selected host cell; or altering the AU composition of a middle portion of the selected mRNA sequence, wherein the middle portion is between the first 35 nucleotides and the last 35 nucleotides of the coding region, to produce an optimized mRNA sequence; and (iii) constructing an mRNA molecule comprising the optimized mRNA sequence.

5. The method of claim 1 , wherein said prokaryotes comprise Escherichia coli (E. coli) bacteria.

6. The method of claim 1 , further comprising constructing a synthetic gene that encodes the optimized mRNA sequence.

7. The method of claim 1 , further comprising producing a protein by expressing a synthetic gene encoding the optimized mRNA sequence in a prokaryotic cell.

8. The method of claim 1 , wherein the selected mRNA sequence is the sequence of a naturally occurring mRNA molecule.

9. The method of claim 1 , wherein altering the AU composition of the last 35nucleotides of the protein coding region of the selected mRNA sequence comprises site directed mutagenesis of a DNA sequence that encodes the selected mRNA sequence.

10. The method of claim 1 , wherein altering the AU composition of the last 35nucleotides of the protein coding region of the selected mRNA sequence comprises synthetic gene assembly.

11. The method of claim 10 , wherein the synthetic gene assembly comprises chemical synthesis.

12. The method of claim 1 , wherein constructing an mRNA molecule comprising the optimized mRNA sequence comprises expressing a synthetic gene encoding the optimized mRNA sequence in a prokaryotic cell.

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