title: Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.
abstract: Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: aymara@fbio.uh.cu.
authors.forename: Aymara
authors.initials: A
authors.lastname: Cabrera-Muñoz
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: laritza@fbio.uh.cu.
authors.forename: Laritza
authors.initials: L
authors.lastname: Rojas
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: gilpradas@gmail.co.
authors.forename: Dayrom F
authors.initials: DF
authors.lastname: Gil
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: ygonzalez64@yahoo.es.
authors.forename: Yamile
authors.initials: Y
authors.lastname: González-González
authors.affiliation: Institut de Biotecnología i de Biomedicina, Universitat Autònoma de Barcelona, Campus Universitari, 08193, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain. Electronic address: mansur_iglesias_manuel@elanco.com.
authors.forename: Manuel
authors.initials: M
authors.lastname: Mansur
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: ayamey07@nauta.cu.
authors.forename: Ayamey
authors.initials: A
authors.lastname: Camejo
authors.affiliation: Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - Bloco E, Sala 10, 21941-902, Rio de Janeiro, RJ, Brazil. Electronic address: jrmpires@cnrmn.bioqmed.ufrj.br.
authors.forename: José R
authors.initials: JR
authors.lastname: Pires
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, Ciudad de La Habana-Cuba, Calle 25 No 455, Vedado, La Habana, Cuba. Electronic address: maday@fbio.uh.cu.
authors.forename: Maday
authors.initials: M
authors.lastname: Alonso-Del-Rivero Antigua
mesh_terms: D000818:Animals; D002417:Cattle; D049851:Gastropoda / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors
keywords: Isotopic labeling; NMR; Pichia pastoris; Recombinant expression; Serine protease inhibitor