title: Conformational and biochemical characterization of a biologically active rat recombinant Protease Nexin-1 expressed in E. coli.
abstract: Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed alpha/beta secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin-rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.
authors.affiliation: Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro "Magna Graecia", Edificio delle Bioscienze, Campus Universitario "Salvatore Venuta", Viale Europa, Germaneto, Catanzaro 88100, Italy. arcone@unicz.it
authors.forename: Rosaria
authors.initials: R
authors.lastname: Arcone
authors.forename: Alberto
authors.initials: A
authors.lastname: Chinali
authors.forename: Nicola
authors.initials: N
authors.lastname: Pozzi
authors.forename: Maddalena
authors.initials: M
authors.lastname: Parafati
authors.forename: Fabio
authors.initials: F
authors.lastname: Maset
authors.forename: Concetta
authors.initials: C
authors.lastname: Pietropaolo
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Filippis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006367:HeLa Cells; D006493:Heparin / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016501:Neurites / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058258:Serpin E2; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D058258:Serpin E2; C541127:Serpine2 protein, rat; D015843:Serpins; D006493:Heparin; D013917:Thrombin