Improved PC1/3 production through recombinant expression in insect cells and larvae.
Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determination of its three-dimensional structure, as well as the understanding of its enzymatic properties, would greatly benefit from the production and availability of large amounts of recombinant enzyme. We report herein improvements in the production of PC1/3 by expressing recombinant mutated forms in both insect cells (Spodoptera frugiperda, Sf9 cells) and larvae (Trichoplusia ni commonly referred to as cabbage looper). On one hand, we deleted the last 135 COOH-terminal residues of mPC1/3 and, on the other hand, we replaced the signal peptide of mPC1/3 by the viral glycoprotein gp67 signal peptide. These modifications were shown to improve markedly (up to 125%) the secretion into the Sf9 cells medium and the amount of enzymatic activity recovered when compared to the original vector. Moreover, intracoelemic injection of the vectors into insect larvae led to the production and purification of enzymatically active enzyme at a level of 30 microg/larva in the case of mPC1/3 and to the production of a high amount of another enzymatically active convertase, PC7. The optimal viral titer for infection of larvae was determined to be 10(6)pfu/ml. Taking into account the purification protocol combined with the ease and efficiency of using larvae, it should now be possible to meet the needs for biochemical and structural studies.
Rabah
Nadia
N
Department of Experimental Medicine, McGill University, Montréal, Que., Canada.
Gauthier
Daniel J
DJ
Gauthier
Dany
D
Lazure
Claude
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors; D006728:Hormones / Q000378:metabolism; D007313:Insecta; D007814:Larva / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D045664:Proprotein Convertase 1 / Q000235:genetics / Q000502:physiology*; D045706:Proprotein Convertase 2 / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D013381:Subtilisins / Q000378:metabolism
D018076:DNA, Complementary; D006728:Hormones; D011994:Recombinant Proteins; C099101:PCSK7 protein, human; D013381:Subtilisins; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2