Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line.
We constructed a recombinant baculovirus, A. californica nuclear polyhedrosis virus, containing the Drosophila Shaker H4 K+ channel cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the cell line Sf9, derived from the army-worm caterpillar S. frugiperda, expresses fully functional Shaker transient K+ currents, as assayed by whole-cell recording. K+ currents begin to appear at about 15 hr after infection, and they continue to increase over the next 3 days. Over the same period of time, a 75 kd band appears on SDS gels stained with Coomassie blue. The identity of this band as a Shaker gene product is confirmed by Western blot analysis using an anti-Shaker antiserum. The 75 kd band accounts for a substantial fraction of the membrane protein in Shaker-infected Sf9 cells. These results give hope that the baculovirus system, which has been used successfully for high-level expression of soluble proteins from higher eukaryotes, may be appropriate for producing large amounts of cloned ion channel proteins as well.
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts.
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K
Klaiber
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Williams
T M
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Roberts
D M
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Papazian
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Jan
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Miller
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000235:genetics; D004553:Electric Conductivity; D005786:Gene Expression Regulation; D007304:Insect Viruses / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D015221:Potassium Channels / Q000378:metabolism*; D011401:Promoter Regions, Genetic
D015221:Potassium Channels; D004247:DNA