Translation Engineering and Synthetic B io lo g y
W. Antoni Kudlicki · 2007
We have developed a toolbox approach for protein engineering to enhance protein expression yield and functionality by manipulating ribosomal “pause” signals correlated with over-represented codon pairs contained in the open reading frame o f genes. C O D A s Translation Engineering algorithms simultaneously optimize each gene design for both organism-specific codon usage and for organism-specific codon pair usage. To further implement Translation Engineering, C O D A has developed propriety gene software, SpeedPlot™, a design tool that predicts and graphically displays the positions o f translational pause sites in genes expressed in native and heterologous biological hosts. For high protein expression levels, SpeedPlot information is used to design Hot-rod™ genes with all predicted pause signals removed for maximal translation elongation rates. For improved protein function, Speedplot information is used to rationally design gene sets using Hot-rod genes as parental templates, from which di rected variant genes are constructed to contain specified combinations o f pause signals (Planned Pause Gene Sets™). Each defined gene set can be tested in parallel for optimal expressors that encode proteins with premium activities. These Translation Engineering methods are enabled by C O D A s proprietary Computationally Optimized D N A Assembly (CO D A) technology to assemble fiill-length genes by thermodynamic necessity. In combination, these technologies make it possible to deliberately alter the predicted translation kinetics patterns o f genes without altering their amino acid sequences. In this chapter, we discuss the application o f these tools for the design and assembly o f synthetic genes optimized for expression and function in any biological host or cell-free protein synthesis system.