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Overcoming codon bias: A method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli

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Abstract

Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein. We have constructed the “RIG” plasmid to overcome the potential codon-bias problem seen in Plasmodium genes. RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes. When co-transformed into E. coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein. Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

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Acknowledgments

We thank James Kane, SmithKline Beecham Pharmaceuticals, for providing pRI952; Alan Cowman, Walter and Eliza Hall Institute for Medical Research (Melbourne, Australia) for providing the anti-DHPS antibody as well as pTrc-PPPK-DHPS from which the pET-DHPS expression system was derived; Ulrich Certa, Roche Pharmaceuticals, for providing the following Plasmodium aldolase expression vectors: p3217, p3255 and p3113; Victoria H. Brophy and Carol H. Sibley, Department of Genetics, University of

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    Citation Excerpt :

    Another approach is the co-transformation of the host with a plasmid that codes for the rare tRNA’s. A number of reports confirm the concept of plasmid mediated tRNA complementation [83–85]. An example of such engineered strains is the E. coli Rosetta strain (Novagen, US) which carries tRNA genes for the rare codons AGG, AGA, AUA, CUA, CCC, and GGA.

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