ABSTRACT
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
Subject(s)
Humans , Metalloendopeptidases/isolation & purification , Pichia/enzymology , Chromatography, Ion Exchange , Metalloendopeptidases/genetics , Pichia/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.
En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.
Subject(s)
Animals , Humans , Rabbits , Vibrio cholerae non-O1/pathogenicity , Argentina/epidemiology , Bacterial Typing Techniques , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/physiology , Chlorocebus aethiops , COS Cells/microbiology , Cholera Toxin/genetics , DNA, Bacterial/genetics , Drug Resistance, Bacterial , Diarrhea/epidemiology , Diarrhea/microbiology , Electrophoresis, Gel, Pulsed-Field , Enterotoxins/genetics , Enterotoxins/isolation & purification , Enterotoxins/physiology , Gene Deletion , Genes, Bacterial , Hemolysin Proteins/genetics , Hemolysin Proteins/isolation & purification , Hemolysin Proteins/physiology , Metalloendopeptidases/genetics , Metalloendopeptidases/isolation & purification , Metalloendopeptidases/physiology , Phylogeny , Vibrio Infections/epidemiology , Vibrio Infections/microbiology , Vibrio cholerae non-O1/drug effects , Vibrio cholerae non-O1/genetics , Vibrio cholerae non-O1/isolation & purification , Virulence/genetics , Water MicrobiologyABSTRACT
Thermostable alkaline metalloprotease (EC 3.4.24.4), a simple protein of very low molecular mass (11 kDa), was isolated and purified from the cell free broth of Streptomyces diastaticus sp SS1 by concentration, (NH4)2SO4 precipitation and affinity chromatography over casein. The isoelectric point of the protease is 7.2. The temperature and pH optima for the enzyme activity are 37 degrees C and 8 respectively. The half life of the enzyme at 55 degrees C is 24 hr and the enzyme is stable over a pH range of 7.5 to 9. The K(m) value of the enzyme was estimated to be 2 x 10(-3) mg ml-1. Ca2+ is essential for enzyme activity as well as thermal stability. The alpha-helix content of the metalloprotease is calculated to be 50% from the circular dichroism spectrum. The number of Zn2+ binding to each molecule of protease is determined to be one by atomic absorption.