Gene Design

Which codon

Codon usage - AGA or CGT - That is the question!

The genetic code provides various codon options for 18 of the 20 amino acids that contribute to the primary sequence of a protein. However, the codon options are used in an unequal frequency in different species showing a clear tendency for certain codons, and it soon became clear that species specific subsets of codons correlate with mRNA expressivity, and thus protein production. It is usually believed that frequently used codons correspond to the most abundant tRNAs pools . Therefore using them avoids tRNA supply shortages, speeds up translation and often dramatically increases heterologous expression.

GC content

Gene expression is regulated by the complex interplay of transcriptional activity, tRNA availability and mRNA stability. The third factor, among others, can be influenced by nucleotide composition, more specifically, GC content. Konu et al. (J Mol Evol, 2002) found that mRNA expression levels were correlated with the presence of G or C at the third nucleotide position of mice and rat codons. CG or atThe influence of overall GC content on expression rates was also observed in insect, (Shieldset al.,1988), yeast (Woo, et al. 2002; Outchkourov et al. 2002; Gurkan et al., 2003), plant (Perlak et al. 1991; Strizhov et al. 1996) and various mammalian genes (Graf et al 2000, Graf et al 2006).

mRNA Structures

Reduce secondary structureIt is commonly accepted that complex and stable mRNA secondary structures may block translational initiation or the translational process (e.g. Wilkstrom et al 1992, Gross et al 1990; Chang et al 1995; ...). Although it is very sophisticated to predict mRNA secondary structures, it is rather easy to disrupt them by avoiding inverted repeat.

Host specific motives

No matter which expression host you are using wildtype sequences are in most cases not adapted for maximum expression. This especially is true for heterologous expression systems. Get rid of Shine Dalgarno sequences, splice sites, poly adenylation signals or any other cis-acting motives within the wildtype open reading frame. Set up a project, define the sequence pattern you want to avoid and minimize occurence of the pattern.The Shine Dalgarno sequence for example (5'-AGGAGGU-3') helps in recruiting the ribosome to the mRNA to initiate protein synthesis. If e.g. a mammalian gene is transfered into E.coli for heterologous protein expression Shine Dalgarno sequences within the wildtyp open reading frame may negatively influence protein expression levels.
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