CODON OPTIMIZATION FOR HIGH-LEVEL EXPRESSION OF HUMAN ERYTHROPOIETIN (EPO) IN MAMMALIAN-CELLS

Codon bias has been observed in many species. The usage of selective c odons in a given gene is positively correlated with its expression eff iciency. As an experimental approach to study codon-usage effects on h eterologous gene expression in mammalian cells, we designed two human erythropoietin(EPO) genes, one in which native codons were systematica lly substituted with codons frequently found in highly expressed human genes and the other with codons prevalent in yeast genes. Relative pe rformances of the re-engineered EPO genes were evaluated with various combinations of promoters and signal leader sequences. Under the compa rable set of combinations, mature EPO gene with human high-frequency c odons gave a considerably higher level of expression than that with ye ast high-frequency codons. However, the levels of EPO expression varie d, depending on the alternate combinations. Since the promoters and th e signal leader sequences that we used are known to be equally efficie nt in gene expression, we hypothesized that the varied expression leve ls were due to the linear sequence between the promoter and the coding gene sequence. To test this possibility, we designed the EPO gene wit h hybrid codon usage in which the 5'-proximal region of the EPO gene w as synthesized with yeast-biased codons and the rest with human-biased codons. This codon-usage hybrid EPO gene substantially enhanced the l evel of EPO transcripts and proteins up to 2.9-fold and 13.8-fold, res pectively, when compared to the revel reached by the original counterp art. Our results suggest that the linear sequence between the promoter and the 5'-proximal region of a gene plays an important role in achie ving high-level expression in mammalian cells. (C) 1997 Elsevier Scien ce B.V.