MersV1, Istex, Corpus, bibRecord, 001554

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

Identifieur interne : 001554 ( Istex/Corpus ); précédent : 001553; suivant : 001555

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

Auteurs : Chrislaine Withers-Martinez ; Elisabeth P. Carpenter ; Fiona Hackett ; Barry Ely ; Mohammed Sajid ; Muni Grainger ; Michael J. Blackman

Source :

Protein Engineering [ 0269-2139 ] ; 1999.

RBID : ISTEX:DFD2A1554CDB710A19F3004531A36C5D6BFA11AD

English descriptors

Teeft :
- Amino acid sequence, Assembly process, Assembly reaction, Baculovirus, Baculovirus system, Blood cells, Blue staining, Cell invasion, Clone, Codon, Codon composition, Codon preference, Codon usage, Codon usage table, Competent cells, Ecorv site, Entire sequence, Error rate, Falciparum, Falciparum gene, Fivetm, Functional screen, Gene, Gene assembly, Gene design, Gene synthesis, Gene synthesis method, Genetics computer group software package, Genome, Heterologous expression, High fivetm cells, High fivetm insect cells, Life technologies, Malaria, Malaria genome, Metal chelate chromatography, Mutation, Mutation frequency, Native gene, Nucleic acids, Nucleotide, Oligos, Outermost primers, Parasite, Pastoris, Pastoris codon usage, Point mutations, Point substitutions, Polyhedrin promoter, Polymerase, Primer, Protein engineering, Protein sequence, Protein structure, Random number generator, Recombinant, Recombinant baculovirus, Recombinant product, Recombinant protein, Sequencing, Single reaction, Subsequent subcloning steps, Synonymous codons, Synthetic gene, Synthetic product, Synthetic sequence, Systematic approach, Unique restriction sites.

Abstract

The A+T-rich genome of the human malaria parasite Plasmodium falciparum encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb Plasmodium falciparum gene (pfsub-1) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR. The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both Pichia pastoris and recombinant baculovirus-infected High FiveTM cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized. Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.

Url:

https://api.istex.fr/ark:/67375/HXZ-D5JNKS6X-M/fulltext.pdf

DOI: 10.1093/protein/12.12.1113

Links to Exploration step

ISTEX:DFD2A1554CDB710A19F3004531A36C5D6BFA11AD

Le document en format XML

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<profileDesc><abstract xml:lang="en"><p>The A+T-rich genome of the human malaria parasite <hi rend="italic">Plasmodium falciparum</hi>
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 gene (<hi rend="italic">pfsub-1</hi>
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      <contrib contrib-type="author"><name><surname>Withers-Martinez</surname>
<given-names>Chrislaine</given-names>
</name>

        

        

      

      <xref rid="AFF1">1</xref>

      <xref rid="AFF2">2</xref>

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<contrib contrib-type="author"><name><surname>Carpenter</surname>
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<given-names>Fiona</given-names>
</name>

        

        

      

      <xref rid="AFF1">1</xref>

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<contrib contrib-type="author"><name><surname>Ely</surname>
<given-names>Barry</given-names>
</name>

        

        

      

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<contrib contrib-type="author"><name><surname>Sajid</surname>
<given-names>Mohammed</given-names>
</name>

        

        

      

      <xref rid="AFF1">1</xref>

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<contrib contrib-type="author"><name><surname>Grainger</surname>
<given-names>Muni</given-names>
</name>

        

        

      

      <xref rid="AFF1">1</xref>

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<contrib contrib-type="author"><name><surname>Blackman</surname>
<given-names>Michael J.</given-names>
</name>

        

        

      

      <xref rid="AFF1">1</xref>

      <xref rid="FN4">4</xref>

      

      

      

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Division of Parasitology, </aff>
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Division of Protein Structure and </aff>
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      <p>The A+T-rich genome of the human malaria parasite <italic>Plasmodium falciparum</italic>
 encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb <italic>Plasmodium falciparum</italic>
 gene (<italic>pfsub-1</italic>
) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR. The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both <italic>Pichia pastoris</italic>
 and recombinant baculovirus-infected High Five<sup>TM</sup>
 cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized. Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.</p>

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PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

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