Dataset Information

Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.

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-? or IL-4 cellular responses were detected between the two antigens.

SUBMITTER: Punde N

PROVIDER: S-EPMC6800875 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.

Punde Neeraja N Kooken Jennifer J Leary Dagmar D Legler Patricia M PM Angov Evelina E

AMB Express 20191019 1

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 pla ...[more]

PMID: 31630257

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Project description:Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Project description:BackgroundNot changing the native constitution of genes prior to their expression by a heterologous host can affect the amount of proteins synthesized as well as their folding, hampering their activity and even cell viability. Over the past decades, several strategies have been developed to optimize the translation of heterologous genes by accommodating the difference in codon usage between species. While there have been a handful of studies assessing various codon optimization strategies, to the best of our knowledge, no research has been performed towards the evaluation and comparison of codon harmonization algorithms. To highlight their importance and encourage meaningful discussion, we compared different open-source codon harmonization tools pertaining to their in silico performance, and we investigated the influence of different gene-specific factors.ResultsIn total, 27 genes were harmonized with four tools toward two different heterologous hosts. The difference in %MinMax values between the harmonized and the original sequences was calculated (ΔMinMax), and statistical analysis of the obtained results was carried out. It became clear that not all tools perform similarly, and the choice of tool should depend on the intended application. Almost all biological factors under investigation (GC content, RNA secondary structures and choice of heterologous host) had a significant influence on the harmonization results and thus must be taken into account. These findings were substantiated using a validation dataset consisting of 8 strategically chosen genes.ConclusionsDue to the size of the dataset, no complex models could be developed. However, this initial study showcases significant differences between the results of various codon harmonization tools. Although more elaborate investigation is needed, it is clear that biological factors such as GC content, RNA secondary structures and heterologous hosts must be taken into account when selecting the codon harmonization tool.

Dataset Information

Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.

Publications

Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity.

Similar Datasets

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