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 valuable tool for the high level production of a multitude of recombinant proteins. However, production of membrane proteins in infected insect cells is often hampered by incorrect folding and processing which results in the accumulation of non-functional protein. Here, we report the construction of a Sf9 insect cell line stably transformed with the ninaA gene from D. melanogaster (Sfn cell line). The ninaA protein is a membrane bound cyclophilin which acts as a peptidyl-prolyl cis/trans isomerase during the folding process of rhodopsin 1 in D. melanogaster rhabdomere. Engineered Sfn insect cells infected with a recombinant baculovirus bearing the human dopamine transporter gene under the control of the polyhedrin promoter showed a > or = 5 times enhanced uptake of [3H]dopamine in comparison to similarly infected Sf9 cells. This increase in specific transport activity was not due to an altered Km 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 results we conclude that the prolyl-cis/trans isomerase activity of the ninaA in the stably transformed Sfn cell line was responsible, directly or indirectly, for the improved folding of the heterologously produced human dopamine transporter.
Lenhard
T
T
Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Frankfurt/M., Germany.
Reiländer
H
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed; D016572:Cyclosporine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics; D006801:Humans; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D011233:Precipitin Tests; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D018411:Spodoptera / Q000166:cytology* / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C068480:ninaA protein, Drosophila; D014415:Tunicamycin; D016572:Cyclosporine; D004298:Dopamine