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Nucleic Acids Research, 2000, Vol. 28, No. 10 2069-2074
© 2000 Oxford University Press

Optimizing heterologous expression in Dictyostelium: importance of 5' codon adaptation

Elisa B. Vervoort, Arno van Ravestein1, Noël N. M. E. van Peij, Judith C. Heikoop1, Peter J. M. van Haastert, Gijs F. Verheijden1 and Maarten H. K. Linskens*

Cell Engineering Facility GBB, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands and 1Research and Development, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-ß gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5–17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium ‘Kozak’-like sequence increased expression levels ~1.5-fold. Thus, using both codon optimization and ‘Kozak’ adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.

* To whom correspondence should be addressed. Tel: +31 50 363 7290; Fax: +31 50 363 4165; Email: m.linskens@chem.rug.nl Present addresses: Noël N. M. E. van Peij, DSM Food Specialties, PO Box 1, 2600 MA Delft, The Netherlands Judith C. Heikoop, McKinsey and Co., Amstel 344, 1017 AS Amsterdam, The Netherlands


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