Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

Zhou, Zhansong; Schnake, Paul; Xiao, Lihua; Lal, Altaf A.

doi:10.1016/j.pep.2003.11.006

Cited by 78 publications

(53 citation statements)

References 42 publications

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“…Moreover, codon usage has been identified as an important factor for efficient gene expression, indicating that differences in the codon usage between two species, in general, and between prokaryotes and eukaryotes, in particular, can result in translation inefficiency (20,27,36,59). Because of this, strategies to remove codons from the native gene that are rare in the new host or the introduction of tRNAs for rare codons into the genome of a given organism have been developed (4,68,71). Another alternative is the complete synthesis of recombinant genes, an option which has become affordable recently, making the adaption of a complete heterologous gene to the codon usage or codon bias of the organism of choice possible.…”

Section: Discussionmentioning

confidence: 99%

Application of the Saccharomyces cerevisiae FLP/ FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

Kopke

Hoff

Kück

2010

Appl Environ Microbiol

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To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single-and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a ⌬Pcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.Site-specific recombination is an important molecular tool for functional genetic studies in both prokaryotes and eukaryotes and is mediated by two major recombinase families, the resolvase/invertase family and the integrase family (9, 65). The resolvase/invertase family is characterized by the conserved catalytic amino acid serine, which allows intramolecular reactions, whereas the integrase family is able to mediate both intra-and intermolecular recombinations due to an autocatalytic activity of the conserved residue tyrosine (19,35). The best-studied members of the integrase family are the integrase of bacteriophage , the recombinase Cre of Escherichia coli bacteriophage P1 (1, 62), the XerCD proteins of E. coli (6), and the eukaryotic FLP recombinase of the yeast Saccharomyces cerevisiae (9, 51). Common to all these systems are two different or identical recognition sites that serve as the DNA substrate for the recombinase.In recent years, recombinases have become important tools to manipulate genetically prokaryotes as well as eukaryotes. In this study, we have modified the FLP/FRT recombination system from yeast for application in filamentous fungi. The FLP/ FRT recombination system is encoded by the 2m (6.4 kb) plasmid that is present in most isolates of S. cerevisiae. D...

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Section: Discussionmentioning

confidence: 99%

Application of the Saccharomyces cerevisiae FLP/ FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

Kopke

Hoff

Kück

2010

Appl Environ Microbiol

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“…The methods used to construct FALVAC genes for expression in E. coli have been detailed previously. 19 Briefly, the amino acid sequence of FALVAC-1A was reverse translated using the program Backtranslation (Entelechon GmbH, Regensburg, Germany) and the gene codons were optimized based on the preferred codon usage of E. coli. Twenty-four overlapping oligonucleotides (50-114 nucleotides in length) coding for the sense and anti-sense DNA strands were chemically synthesized at CDC's Biotechnology Core Facility.…”

Section: Methodsmentioning

confidence: 99%

Development, Characterization and Immunogenicity of a Multi-Stage, MultivalentPlasmodium falciparumVaccine Antigen (FALVAC-1A) Expressed inEscherichia coli

Zhou¹,

Todd²,

Wohlhueter³

et al. 2006

Human Vaccines

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A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.

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“…The excessive presence of rare codons can result in ribosome stalling, slow translation, and translation errors. In some cases, rare codons inhibit protein synthesis and cell growth [55]. Several approaches have been used successfully to compound these problems.…”

Section: Discussionmentioning

confidence: 99%

GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine

Döşkaya

Kalantari-Dehaghi

Walsh

et al. 2007

Vaccine

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The present study evaluates immunogenicity and protection potency of a codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the three GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine.

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Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

Cited by 78 publications

References 42 publications

Application of the Saccharomyces cerevisiae FLP/ FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

Application of the Saccharomyces cerevisiae FLP/ FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

Development, Characterization and Immunogenicity of a Multi-Stage, MultivalentPlasmodium falciparumVaccine Antigen (FALVAC-1A) Expressed inEscherichia coli

GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine

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