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 protei...

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Bibliographic Details
Published in:Biotechnology techniques 1999-07, Vol.13 (7), p.485-489
Main Authors: NORAZMI, M. N, ZAINUDDIN, Z. F, SUPPIAN, R, DALE, J. W
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Fundamental and applied biological sciences. Psychology
Health. Pharmaceutical industry
Industrial applications and implications. Economical aspects
Mycobacterium smegmatis
Plasmodium falciparum
Production of active biomolecules
Vaccins
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Summary: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 super(4) c.f.u. mu g super(-1) DNA) and a substantial increase in the expression of the recombinant protein.
ISSN:0951-208X
1573-6784
DOI:10.1023/A:1008945112490