ENGINEERING THE FOLDING PATHWAY OF INSECT CELLS - GENERATION OF A STABLY TRANSFORMED INSECT-CELL LINE SHOWING IMPROVED FOLDING OF A RECOMBINANT MEMBRANE-PROTEIN

The baculovirus-insect cell expression system has proven to be a valua ble tool for the high level production of a multitude of recombinant p roteins. However, production of membrane proteins in infected insect c ells is often hampered by incorrect folding and processing which resul ts in the accumulation of non-functional protein. Here, we report the construction of a Sf9 insect cell line stably transformed with the nin aA gene from D. melanogaster (Sfn cell line). The ninaA protein is a m embrane bound cyclophilin which acts as a peptidyl-prolyl cis/trans is omerase during the folding process of rhodopsin 1 in D. melanogaster r habdomere. Engineered Sfn insect cells infected with a recombinant bac ulovirus bearing the human dopamine transporter gene under the control of the polyhedrin promoter showed a greater than or equal to 5 times enhanced uptake of [H-3]dopamine in comparison to similarly infected S f9 cells. This increase in specific transport activity was not due to an altered K-m value in the Sfn cell line. The uptake in infected Sfn cells was blocked by the peptidyl-prolyl cis/trans isomerase inhibitor cyclosporin A which had no effect on infected Sf9 cells. From these r esults we conclude that the prolyl cis/trans isomerase activity of the ninaA in the stably transformed Sm cell line was responsible, directl y or indirectly, for the improved folding of the heterologously produc ed human dopamine transporter. (C) 1997 Academic Press.