Design Parameters to Control Synthetic Gene Expression in Escherichia coli

Abstract

BackgroundProduction of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles.Principal FindingsTo identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well.ConclusionThe systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…However, during starvation the isoacceptors for the rate-limiting amino acid were selectively charged, confirming the theoretical predictions of Elf et al (Dittmar et al 2005). Thus, the theoretical model correctly identifies and orders the amino acid starvation-insensitive isoacceptors and based on Welch et al results (Welch, Govindarajan, et al 2009), this might be an important design criterion for genes for heterologous protein expression.…”

“…This may place strain on the cell and hence, the principles of codon bias for heterologous protein expression may differ substantially from those for endogenous expression. Thus, codons that are preferred for endogenous gene expression may not be the same as those for heterologous gene expression (Fath et al 2011;Plotkin & Kudla 2011; (Welch, Govindarajan, et al 2009). They measured the levels of protein expression and determined that it varied 40-fold between the highest and lowest expressing genes.…”

“…Thus, they hypothesized that as amino acid charging of tRNA becomes limiting, only the tRNAs that are robust to starvation can support high translation levels of heterologous protein expression. Therefore successful codon bias of a gene depends on maintaining high levels of charged tRNAs and minimizing levels of uncharged tRNAs which can inhibit translation (Bonomo & Gill 2005;Dong et al 1995;Harcum 2002;Welch, Govindarajan, et al 2009). …”

Strategies for production of active eukaryotic proteins in bacterial expression system

“…Codon bias becomes highly prevalent problems when rare codons in the transcripts form clusters such as doublets or triplets accumulation that is large in quantities. Translation error arised from rare codon bias includes mistranslational amino acid substitutions, frameshifting events or premature translational termination [2][3][4] .…”

“…Higher concentration of the unfolded protein often leads to decreased refolding yields, regardless of refolding method. So, it is desirable to keep the concentration of the initial un-folded protein to a minimum level if higher and correct refolding proteins are expected [1,3] .…”