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.
Klaiber
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K
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts.
Williams
N
N
Roberts
T M
TM
Papazian
D M
DM
Jan
L Y
LY
Miller
C
C
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