Abstract

Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

Highlights

  • Escherichia coli expression systems have been widely used for the expression and manufacturing of various malarial antigens owing to their ease of use and advantages in cost and scale despite protein expression and folding obstacles

  • We developed a recombinant protein vaccine candidate based on PfCelTOS in E. coli

  • A Histidine tag-free PfCelTOS was expressed in E. coli as above, and similar to the N- and CH-PfCelTOS proteins were expressed without the native PfCelTOS signal sequence

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Summary

Introduction

Escherichia coli expression systems have been widely used for the expression and manufacturing of various malarial antigens owing to their ease of use and advantages in cost and scale despite protein expression and folding obstacles. More frequently used codons are often found in well-ordered structural elements such as alpha helices, while low usage frequency codons often occur within link/end segments (Thanaraj and Argos 1996). These observations suggest that codon usage frequency plays an inherent role in cotranslational folding. Based on these concepts, we developed a strategy to “recode” target gene sequences for heterologous expression by substituting native codons with synonymous alternates with identical or similar usage frequencies in the expression host. Putative link/end segments are identified and recoded to re-establish regions benefitted by slower translation (Angov et al 2008)

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