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 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 Five(TM) 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.
Withers-Martinez
C
C
Division of Parasitology, Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Carpenter
E P
EP
Hackett
F
F
Ely
B
B
Sajid
M
M
Grainger
M
M
Blackman
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D016054:DNA, Protozoan / Q000138:chemical synthesis; D004587:Electrophoresis, Agar Gel; D017125:Genes, Protozoan / Q000235:genetics; D016678:Genome; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia; D010963:Plasmodium falciparum / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000235:genetics; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013381:Subtilisins; C403176:subtilisin-like protease 1, Plasmodium falciparum