The use of assembly polymerase chain reaction for cloning of a malarial epitope into Mycobacterium smegmatis -: importance of overcoming codon bias

The cloning of an A:T-rich DNA fragment, the C-terminus of Plasmodium falciparum merozoite surface protein-1, into the G:C-rich Mycobacterium smegmatis resulted in delayed growth (6 days), a 10-fold decrease in the expected transformation efficiency and low level expression of the recombinant protein by the host cells. The technique of assembly PCR was used to assemble a series of oligonucleotides to generate a synthetic homologue of the plasmodium DNA fragment using mycobacterium codon bias. Cloning of the synthetic fragment into M. smegmatis resulted in normal growth (4 days), the expected level of transformation efficiency (10(4) c.f.u. mu g(-1) DNA) and a substantial increase in the expression of the recombinant protein.