Molecular Characterization, Enzymatic Analysis, and Purification of Murine Proprotein Convertase-1/3 (PC1/PC3) Secreted from Recombinant Baculovirus-Infected Insect Cells
| Autor: | Diane Savaria, Alain Boudreault, Claude Lazure, Nabil G. Seidah, Normand Rondeau, Michel Chrétien, Dany Gauthier |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
1-Deoxynojirimycin
DNA Complementary Glycosylation Recombinant Fusion Proteins Genetic Vectors Vaccinia virus Proprotein convertase 1 CHO Cells Spodoptera Biology Cell Line law.invention Mice chemistry.chemical_compound Cricetulus law Cricetinae Animals Aspartic Acid Endopeptidases Secretion Enzyme Inhibitors Proprotein Molecular mass Sulfates Swainsonine Tunicamycin Glycosyltransferases Hydrogen-Ion Concentration Molecular biology Nucleopolyhedroviruses Proprotein Convertase 1 Biochemistry chemistry Cell culture Zymogen activation Recombinant DNA Calcium Proprotein Convertases Protein Processing Post-Translational Biotechnology |
| Zdroj: | Protein Expression and Purification. 14:353-366 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.1998.0964 |
| Popis: | A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH 2 -terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated. |
| Databáze: | OpenAIRE |
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