The production of an active protein kinase C-delta in insect cells is greatly enhanced by the use of the basic protein promoter.
Occasionally, only a small percentage of recombinant proteins produced in the baculovirus expression system are functionally active. We had previously shown that the majority of protein kinase C-delta (PKC-delta) produced in insect cells was inactive; less than 1% of the expressed enzyme had lipid-dependent kinase activity. In this report, we have attempted to optimize the production of a catalytically active PKC-delta. Under optimum conditions, we were able to increase the levels of PKC-delta from 10-20% to about 65% of the total cellular protein; however, there was no increase in the levels of catalytically active enzyme. Expression of PKC-delta as a fusion protein or as a secreted protein also met with limited success. Under all conditions, expression of PKC-delta proteins under control of the strong polyhedrin promoter resulted in the production of large amounts of inactive enzyme. Expression under the control of the basic protein promoter, Pcor, resulted in the reduction of the levels of recombinant protein by a factor of about four, but the PKC-delta enzyme produced under these conditions was 10- to 15-fold more active. Thus, the earlier temporal expression of PKC-delta in insect cells resulted in the production of more active enzyme.
Rankl
N B
NB
Sphinx Pharmaceuticals Corporation, Molecular Biology Section, Durham, North Carolina 27717.
Rice
J W
JW
Gurganus
T M
TM
Barbee
J L
JL
Burns
D J
DJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005822:Genetic Vectors; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D000077183:Occlusion Body Matrix Proteins; D010703:Phorbol Esters / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D051745:Protein Kinase C-delta; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D007527:Isoenzymes; D000077183:Occlusion Body Matrix Proteins; D010703:Phorbol Esters; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D011493:Protein Kinase C; D051745:Protein Kinase C-delta