Codon usage: nature's roadmap to expression and folding of proteins

Abstract

Biomedical and biotechnological research relies on processes leading to the successful expression and production of key biological products. High-quality proteins are required for many purposes, including protein structural and functional studies. Protein expression is the culmination of multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and post-translational modifications leading to the formation of a stable product. Although significant strides have been achieved over the past decade, advances toward integrating genomic and proteomic information are essential, and until such time, many target genes and their products may not be fully realized. Thus, the focus of this review is to provide some experimental support and a brief overview of how codon usage bias has evolved relative to regulating gene expression levels.

Figure 1

Codon harmonization improves expression level of P. falciparum malaria protein, MSP1₄₂ in E. coli. (A) Coomassie Blue-stained gel on total cell lysates, uninduced (U) and induced (I) cells (3 h with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG)), expressing P. falciparum MSP1₄₂ from various constructs. (B) Western blot on total cell lysates (same as above) probed with rabbit polyclonal anti-MSP1₄₂ antibodies for U and I cells (3 h with 0.1 mM IPTG), expressing P. falciparum MSP1₄₂ from various constructs. Lane 1: MSP1₄₂ with a single, synonymous codon substitution; lane 2: MSP1₄₂ codon-harmonized at the 5′-end, first thirty codons; and lane 3: MSP1₄₂ full gene sequence codon-harmonized. Arrow points to the expressed protein band.

Figure 2

Quantitation of expression levels of P. falciparum MSP1₄₂ following partial purification. Cells expressing various P. falciparum MSP1₄₂ constructs were purified by nickel-affinity chromatography. Construct A represents the yield from the native gene sequence of P. falciparum MSP1₄₂. Construct B represents the yield from the single, synonymous codon substitution (pause-site mutant). Construct C represents the yield from the 5′-end first thirty harmonized codons. Construct D represents the yield from the full gene codon-harmonized sequence. Arrow points to the partially purified band.

Figure 3

Schematic model of co-translational folding on mRNA by ribosomes. (A) Ribosomal complex centered on the translation initiation site, AUG (initiation codon). (B) Nascent polypeptide synthesis within the protective environment of the ribosomal tunnel. (C) Putative translational pause sites in conjunction with co-translational folding occur within the ribosomal tunnel. Differences in codon usage frequency are shown as thick dashed lines with arrowheads for areas representing high-frequency-usage codons, and therefore, translating rapidly (hare) and regions that are double lined represent segments of lower frequency usage codons (i.e., putative pause sites; tortoise) where translation proceeds more slowly to allow nascent polypeptide folding.