Modeling ribosome dynamics to optimize heterologous protein production

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.

1 . A method for predicting 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; and   (b) determining a total cycle count across codons throughout the coding region;   
       wherein the total cycle count across codons throughout the coding region is correlated with protein yield. 
     
     
         2 . A method for predicting 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;   
       wherein the maximum sum in the translation bottleneck plot is correlated with protein yield. 
     
     
         3 . (canceled) 
     
     
         4 . A method for increasing protein yield for translation of a gene, the method comprising:
 (a) performing the method for predicting protein yield for translation of a gene of  claim 1 ; and   (b) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time;   
       wherein the protein yield for translation of the gene is increased. 
     
     
         5 . The method of  claim 4 , wherein step 4(b) comprises modifying codons such that ribosome wait time is decreased. 
     
     
         6 . The method of  claim 4 , 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. 
     
     
         7 . A method for decreasing protein yield for translation of a gene, the method comprising:
 (a) performing the method for predicting protein yield for translation of a gene of  claim 1 ; and   (b) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time;   
       wherein the protein yield for translation of the gene is decreased. 
     
     
         8 . The method of  claim 7 , wherein step 7(b) comprises modifying codons such that ribosome wait time is increased. 
     
     
         9 . A method for predicting protein aggregation, 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; and   (b) determining a total cycle count across codons throughout the coding region;   
       wherein the total cycle count across codons throughout the coding region is correlated with protein aggregation. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . A method for increasing protein aggregation, the method comprising:
 (a) performing the method for predicting protein aggregation of  claim 9 ; and   (b) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time;   
       wherein protein aggregation is increased. 
     
     
         13 . The method of  claim 12 , wherein step 12(b) comprises modifying codons such that ribosome wait time is decreased. 
     
     
         14 . A method for decreasing protein aggregation, the method comprising:
 (a) performing the method for predicting protein aggregation of  claim 9 ; and   (b) modifying codons using synonymous codons that conserve the protein amino acid sequence while changing the force and/or the wait time;   
       wherein protein aggregation is decreased. 
     
     
         15 . The method of  claim 14 , wherein step 14(b) comprises modifying codons such that ribosome wait time is increased. 
     
     
         16 . The method of  claim 1 , wherein the gene is from a prokaryotic organism and the 3′ terminal rRNA tail of the ribosome is an 16S rRNA tail. 
     
     
         17 . The method of  claim 1 , wherein the gene is from a eukaryotic organism and the 3′ terminal rRNA tail of the ribosome is an 18S rRNA tail. 
     
     
         18 . A computer readable medium programmed to perform one or more of the method steps of  claim 1 .