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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.
AMB Express 2019; 9(1):167AE

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.

Authors+Show Affiliations

Malaria Biologics Branch, Walter Reed Army Institute of Research, Silver Spring, MD, 20910, USA. The Geneva Foundation, 917 Pacific Avenue, Tacoma, WA, 98402, USA.Malaria Biologics Branch, Walter Reed Army Institute of Research, Silver Spring, MD, 20910, USA.Naval Research Laboratories, 4555 Overlook Ave, Washington, DC, 20375, USA.Naval Research Laboratories, 4555 Overlook Ave, Washington, DC, 20375, USA.Malaria Biologics Branch, Walter Reed Army Institute of Research, Silver Spring, MD, 20910, USA. evelina.angov.civ@mail.mil.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31630257

Citation

Punde, Neeraja, et al. "Codon Harmonization Reduces Amino Acid Misincorporation in Bacterially Expressed P. Falciparum Proteins and Improves Their Immunogenicity." AMB Express, vol. 9, no. 1, 2019, p. 167.
Punde N, Kooken J, Leary D, et al. Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity. AMB Express. 2019;9(1):167.
Punde, N., Kooken, J., Leary, D., Legler, P. M., & Angov, E. (2019). Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity. AMB Express, 9(1), p. 167. doi:10.1186/s13568-019-0890-6.
Punde N, et al. Codon Harmonization Reduces Amino Acid Misincorporation in Bacterially Expressed P. Falciparum Proteins and Improves Their Immunogenicity. AMB Express. 2019 Oct 19;9(1):167. PubMed PMID: 31630257.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity. AU - Punde,Neeraja, AU - Kooken,Jennifer, AU - Leary,Dagmar, AU - Legler,Patricia M, AU - Angov,Evelina, Y1 - 2019/10/19/ PY - 2019/09/17/received PY - 2019/10/01/accepted PY - 2019/10/21/entrez PY - 2019/10/21/pubmed PY - 2019/10/21/medline KW - Alpha-helical content KW - Amino-acid misincorporation KW - Codon-harmonization KW - PfCelTOS KW - Protein translation KW - Secondary structure SP - 167 EP - 167 JF - AMB Express JO - AMB Express VL - 9 IS - 1 N2 - 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. SN - 2191-0855 UR - https://www.unboundmedicine.com/medline/citation/31630257/Codon_harmonization_reduces_amino_acid_misincorporation_in_bacterially_expressed_P._falciparum_proteins_and_improves_their_immunogenicity L2 - https://dx.doi.org/10.1186/s13568-019-0890-6 DB - PRIME DP - Unbound Medicine ER -