Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host

Abstract

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.

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

“…This means the gene sequence still This article is protected by copyright. All rights reserved 14 retains its preferred and rare codons, these are just altered to the equivalent preferred and rare synonymous codons used by the host organism (Angov et al 2008;Angov et al 2011;Angov 2011). The codon harmonization algorithm was applied to genes encoding three Plasmodium falciparum proteins to be expressed in E. coli and gene expression was subsequently determined to increase from 4 to 1,000 fold that of non-optimized genes (Angov et al 2008).…”

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

“…Codon harmonization (Angov et al, 2008;Angov, 2011;Angov et al, 2011) is thought to assist protein folding in heterologous host systems by mimicking the translation rates of the native host in a heterologous strain. The translation rate is determined by codon usage frequencies, where the presence of infrequently used codons forces a reduction of the translation rate, allowing evolving protein to fold in phases (Angov et al, 2008;Angov, 2011;Angov et al, 2011). Both the frequency and positioning of infrequently used codons is critical for protein folding.…”

“…The principal of codon harmonization (Angov et al, 2008) is to substitute synonymous codons from the heterologous host such that the codon usage frequency, positioning and therefore rhythm of translation follows that of the native host. In order to codon harmonize the GoxPDC gene for G. thermoglucosidasius expression, codon substitutions were made to the wild type gene sequence so as to match the usage frequencies found in the native host for each codon position while maintaining the amino acid composition of the wild-type protein.…”

“…thermoglucosidasius, without the need to calculate link / end segments (Thanaraj et al, 1996;Angov et al, 2008).…”

“…It has been demonstrated that the position and usage frequency of codons, together, play a role in correct protein folding and that "codon harmonization" could be used to overcome poor expression, at least in E. coli (Angov et al, 2008;Rosano et al, 2009). Incompatibilities in codon usage and their effect on expression of PDCs have been reported (Lowe et al, 1992;Talarico et al, 2001;Talarico et al, 2005).…”