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

Expression of heterologous proteins in Dictyostelium discoideum presents un ique research opportunities, such as the functional analysis of complex hum an glycoproteins after random mutagenesis, In one study, human chorionic go nadotropin (hCG) and human follicle stimulating hormone were expressed in D ictyostelium, During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start c odon, The Dictyostelium genome has a higher AT content than the human, resu lting in a different codon preference. The hCG-beta gene contains three clu sters with infrequently used codons that were changed to codons that are pr eferred by Dictyostelium, The results reported here show that optimizing th e first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased e xpression 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 p hase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence Increased expression levels simi lar to 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation , a 6- to El-fold increase expression levels could be obtained for hCG.