    4.2.2.20 | more |
engineering of a mammalian expression system of an epitope-tagged chondroitinase ABC protein. At the 5'-end, an optimized KOZAK-ATG sequence is inserted, to allow efficient eukaryotic translation, followed by a 60 base pair secretion sequence from the fish Oreochromis aureus, engineered for efficient crosshost recombinant protein expression. At the 3'-end, a 3xFlag epitope tag is added. The addition of the eukaryotic secretion signal sequence allows secretion, but interferes with function of the secreted enzyme. In contrast, expression of the chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence leads to efficient secretion of a biologically active chondroitinase ABC protein from both immortalized and primary cells. The C-terminal epitope tag can be utilized to follow expression of this protein |
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