title: High-level expression in Escherichia coli, purification and kinetic characterization of Plasmodium falciparum M1-aminopeptidase.
abstract: Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1mM IPTG at 37°C for 18h. This allowed obtaining 100mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn(2+) excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors.
authors.affiliation: Centro de Estudio de Proteínas, 25 # 455 entre J e I, Facultad de Biología, Universidad de La Habana, Cuba. Electronic address: jogoba@fbio.uh.cu.
authors.forename: Jorge
authors.initials: J
authors.lastname: González-Bacerio
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Ave. Universidad 2001, Cuernavaca, Mor., Mexico. Electronic address: joel@ibt.unam.mx.
authors.forename: Joel
authors.initials: J
authors.lastname: Osuna
authors.affiliation: Centro de Estudio de Proteínas, 25 # 455 entre J e I, Facultad de Biología, Universidad de La Habana, Cuba. Electronic address: aponce@estudiantes.fbio.uh.cu.
authors.forename: Amaia
authors.initials: A
authors.lastname: Ponce
authors.affiliation: Centro Nacional de Investigaciones Científicas, Ave. 25 y 158, 12100 La Habana, Cuba. Electronic address: rafael.fando@cnic.edu.cu.
authors.forename: Rafael
authors.initials: R
authors.lastname: Fando
authors.affiliation: Fundación Instituto de Estudios Avanzados, Carretera Nacional de Hoyo de la Puerta, Valle de Sartenejas, Baruta 1080, Estado Miranda, Venezuela. Electronic address: kfigarella@idea.gob.ve.
authors.forename: Katherine
authors.initials: K
authors.lastname: Figarella
authors.affiliation: Centro de Estudio de Proteínas, 25 # 455 entre J e I, Facultad de Biología, Universidad de La Habana, Cuba. Electronic address: yanira@quimica.uh.cu.
authors.forename: Yanira
authors.initials: Y
authors.lastname: Méndez
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Ave. Universidad 2001, Cuernavaca, Mor., Mexico. Electronic address: charli@ibt.unam.mx.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Charli
authors.affiliation: Centro de Estudio de Proteínas, 25 # 455 entre J e I, Facultad de Biología, Universidad de La Habana, Cuba. Electronic address: mchavez@infomed.sld.cu.
authors.forename: María de Los Á
authors.initials: Mde L
authors.lastname: Chávez
mesh_terms: D000626:Aminopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D000626:Aminopeptidases
keywords: Expression in Escherichia coli; IMAC; M1-family aminopeptidase; Malaria; PfA-M1; Plasmodium falciparum