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

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...

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Bibliographic Details
Published in:Protein engineering 1999-12, Vol.12 (12), p.1113-1120
Main Authors: Withers-Martinez, Chrislaine, Carpenter, Elisabeth P., Hackett, Fiona, Ely, Barry, Sajid, Mohammed, Grainger, Muni, Blackman, Michael J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Baculoviridae
Base Sequence
DNA, Protozoan - chemical synthesis
Electrophoresis, Agar Gel
gene synthesis
Genes, Protozoan - genetics
Genome
Humans
Molecular Sequence Data
pfsub-1 gene
Pichia
Pichia pastoris
Plasmodium falciparum
Plasmodium falciparum - genetics
Polymerase Chain Reaction - methods
protein expression
Protozoan Proteins
Recombinant Proteins - genetics
subtilisin-like protease
Subtilisins - biosynthesis
Subtilisins - genetics
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Summary: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.
ISSN:0269-2139
1741-0126
1460-213X
1741-0134
DOI:10.1093/protein/12.12.1113