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US 2020/0286578 A1Published application
Modeling ribosome dynamics to optimize heterologous protein production
AssigneeNorth Carolina State University
InventorsScott Khoi Vu, Donald L. Bitzer
Life of this patent
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Abstract
The presently disclosed subject matter provides a free-energy based model of translation elongation to predict and optimize heterologous gene expression. The model and software allow for the prediction and optimization of genes for increased or decreased protein yield and for increased or decreased protein aggregation.
Claims7
A method for increasing protein yield for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) determining a total cycle count across codons throughout the coding region;
- and
- (c) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time, and wherein the ribosome displacement magnitude is minimized by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein yield is increased.
2 more elements
A method for increasing protein aggregation for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) determining a total cycle count across codons throughout the coding region;
- and
- (c) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time, and wherein the ribosome displacement magnitude is minimized by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein aggregation is increased.
2 more elements
A method for increasing protein yield for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) plotting a translation bottleneck plot, wherein the plot comprises values comprising a sum of cycles within a sliding window of size N codons;
- and
- (c) determining a maximum sum in the translation bottleneck plot, and wherein the ribosome displacement magnitude is minimized by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein yield is increased.
2 more elements
A method for increasing protein aggregation for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) plotting a translation bottleneck plot, wherein the plot comprises values comprising a sum of cycles within a sliding window of size N codons;
- and
- (c) determining a maximum sum in the translation bottleneck plot, and wherein the ribosome displacement magnitude is minimized by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein aggregation is increased.
2 more elements
A method for increasing protein yield for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) performing a protein translation simulation to determine an amount of ribosomes that finish translation within a number of cycles, wherein the amount of ribosomes that finish translation within a number of cycles is correlated with protein yield;
- and
- (c) minimizing the ribosome displacement magnitude by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein yield is increased.
2 more elements
A method for increasing protein aggregation for translation of a gene, the method comprising:
- (a) determining ribosome wait time at each codon in a coding region of an mRNA encoding the protein, comprising determining a number of cycles at each codon, wherein the number of cycles is a function of tRNA abundance, ribosome displacement magnitude, and force from binding between the mRNA and a 3′ terminal rRNA tail of the ribosome;
- (b) performing a protein translation simulation to determine an amount of ribosomes that finish translation within a number of cycles, wherein the amount of ribosomes that finish translation within a number of cycles is correlated with protein aggregation;
- and
- (c) minimizing the ribosome displacement magnitude by selecting for a phase angle of the gene that is substantially equal to a species angle of the gene;
- wherein the protein aggregation is increased.
2 more elements
7-18. (canceled)
Dependencies are inferred from the claim text.