“…). It is important to note that these results differ from previous studies of production of recombinant IDS in P. pastoris at standard conditions , which showed higher enzyme activity levels than those reported in the present study. However, differences in substrate and enzyme activity assay conditions, limit the comparison among these studies.…”
Section: Resultscontrasting
confidence: 99%
“…Equivalent volumes of purified recombinant proteins were loaded and run on a 12% (w/v) SDS–PAGE and electroblotted onto nitrocellulose membranes (Hybond C‐Extra; Amersham Bioscience, Piscataway, NJ, USA). Membranes were blocked with a blocking buffer containing PBS 1× (composition per liter: 8 g NaCl, 0.2 g KCl, 1.44 g NaH 2 PO 4 , 0.24 g KH 2 PO 4 , pH 7.2 ± 0.2), 0.3% (v/v) Tween 20, and 5% (w/v) BSA (Sigma‐Aldrich, St. Louis, MO, USA) at room temperature for 1 H. After blocking, all membranes were incubated overnight at 4 °C with primary chicken IgY anti‐human IDS (1:500 in blocking buffer) , followed by incubation with a peroxidase‐conjugated goat anti‐chicken (1:2,000, Promega, Madison, WI, USA) for 1 H at room temperature. The specific bands were visualized using enhanced chemiluminescence (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Fisher Scientific).…”
Section: Methodsmentioning
confidence: 99%
“…As a continuation on the development of an ERT for MPS II using an enzyme produced in P. pastoris , in this study we evaluated the effect of culture conditions (i.e., limited‐oxygen and limited‐substrate conditions) and codon optimization on the production of rIDS in P. pastoris (prIDS). Furthermore, the temperature pH and serum stability, and the cell uptake and intracellular trafficking for prIDS, were also evaluated.…”
Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg H and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.
“…). It is important to note that these results differ from previous studies of production of recombinant IDS in P. pastoris at standard conditions , which showed higher enzyme activity levels than those reported in the present study. However, differences in substrate and enzyme activity assay conditions, limit the comparison among these studies.…”
Section: Resultscontrasting
confidence: 99%
“…Equivalent volumes of purified recombinant proteins were loaded and run on a 12% (w/v) SDS–PAGE and electroblotted onto nitrocellulose membranes (Hybond C‐Extra; Amersham Bioscience, Piscataway, NJ, USA). Membranes were blocked with a blocking buffer containing PBS 1× (composition per liter: 8 g NaCl, 0.2 g KCl, 1.44 g NaH 2 PO 4 , 0.24 g KH 2 PO 4 , pH 7.2 ± 0.2), 0.3% (v/v) Tween 20, and 5% (w/v) BSA (Sigma‐Aldrich, St. Louis, MO, USA) at room temperature for 1 H. After blocking, all membranes were incubated overnight at 4 °C with primary chicken IgY anti‐human IDS (1:500 in blocking buffer) , followed by incubation with a peroxidase‐conjugated goat anti‐chicken (1:2,000, Promega, Madison, WI, USA) for 1 H at room temperature. The specific bands were visualized using enhanced chemiluminescence (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Fisher Scientific).…”
Section: Methodsmentioning
confidence: 99%
“…As a continuation on the development of an ERT for MPS II using an enzyme produced in P. pastoris , in this study we evaluated the effect of culture conditions (i.e., limited‐oxygen and limited‐substrate conditions) and codon optimization on the production of rIDS in P. pastoris (prIDS). Furthermore, the temperature pH and serum stability, and the cell uptake and intracellular trafficking for prIDS, were also evaluated.…”
Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg H and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.
“…Under these conditions, the recombinant enzyme was found extracellularly with an activity of 29.5 nmol h −1 mg −1 . In spite of the presence of the pre-pro signal sequence of the alpha mating factor, the recombinant enzyme was also found intracellularly in soluble and aggregated forms ( [57,58] and unpublished data). Finally, a P. pastoris-codon optimized version of the human IDS cDNA was used to increase the production of the enzyme, which allowed an increase in enzyme activity, reaching up to 49.7 nmol h −1 mg −1 (unpublished data).…”
“…3 ) allowing the understanding, correlation and prediction of phenotype-genotype correlations, as well as docking and molecular dynamic modeling against natural and artificial substrates [ 90 – 92 ]. Structural modelling of IDS allowed the identification and design of peptides to produce chicken immunoglobulin Y (IgY) anti-IDS antibodies, which were used for the development of an ELISA test [ 93 ]. Fig.…”
The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
