Optimization of codon pair use within the (GGGGS)3 linker sequence results in enhanced protein expression

Here, we report that a significant increase in recombinant fusion antibody expression can be accomplished by adjusting the nucleotide sequence to conform to certain codon pairing rules. We investigated the expression of a protein in which a single chain Fv specific for HER2/neu with VH and VL joined by a flexible (GGGGS)3 linker was linked to the CH3 of a human anti-rat transferrin receptor IgG3 heavy chain with the same flexible (GGGGS)3 linker. In initial experiments we failed to achieve significant expression of this protein. However, when we made a single nucleotide change in each (GGGGS)3 linker we were able to achieve expression The change of one nucleotide within each linker did not alter either the amino acid sequence or the frequency score of these codon triplets' usage in mammalian cells. Instead they removed two codon pairs predicted to be detrimental to expression. In a transient transfection assay we find that this change results in an over 30-fold increase in expression that is not the result of an increase in the level of accumulated mRNA. In addition, the changes made it possible to isolate stably transfected mammalian cell clones producing high levels of fusion protein, which had not been possible using the original gene.