חיפוש מתקדם
Federici, C., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Eshdat, Y., Dept. Fruit Tree Breed. Molec. G., Agricultural Research Organization, Volcani Center, 50250, Bet-Dagan, Israel
Richard, I., URA 1922 CNRS, Généthon, 1 rue de l'Internationale, 91002, Evry, France
Bertin, B., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Guillaume, J.L., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Hattab, M., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Beckmann, J.S., URA 1922 CNRS, Généthon, 1 rue de l'Internationale, 91002, Evry, France
Strosberg, A.D., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Camoin, L., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS- PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30- 31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme.
פותח על ידי קלירמאש פתרונות בע"מ -
הספר "אוצר וולקני"
אודות
תנאי שימוש
Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems
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Federici, C., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Eshdat, Y., Dept. Fruit Tree Breed. Molec. G., Agricultural Research Organization, Volcani Center, 50250, Bet-Dagan, Israel
Richard, I., URA 1922 CNRS, Généthon, 1 rue de l'Internationale, 91002, Evry, France
Bertin, B., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Guillaume, J.L., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Hattab, M., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Beckmann, J.S., URA 1922 CNRS, Généthon, 1 rue de l'Internationale, 91002, Evry, France
Strosberg, A.D., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Camoin, L., Inst. Cochin de Genet. Molec., Lab. d'Immunopharmacologie Molec., CNRS UPR 415, 22 rue Méchain, 75014, Paris, France
Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems
Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS- PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30- 31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme.
Scientific Publication