5 research outputs found
Human sulfatase transiently and functionally active expressed in E. coli K12
The recombinant human iduronate 2-sulfate sulfatase (hrIDS) was
transiently and functionally active expressed in E. coli K12. The
enzyme activity (crude extract) at 100 ml and 400 ml oscillated between
0.25 and 10.58 nmol h-1 mg-1. The wide Western-blot peptide profile
suggest that hrIDS is proteolitically processed \u201crandomly\u201d
which agrees with the ultrafiltration assay in which the hrIDS activity
was found in all fractions (<30kDa, 30-100kDa and >100kDa). No
glycation sites were found by computer analysis of the hIDS sequence;
discarding the possibility of marks for glycation and proteolytic
processing
Human sulfatase transiently and functionally active expressed in E. coli K12
The recombinant human iduronate 2-sulfate sulfatase (hrIDS) was
transiently and functionally active expressed in E. coli K12. The
enzyme activity (crude extract) at 100 ml and 400 ml oscillated between
0.25 and 10.58 nmol h-1 mg-1. The wide Western-blot peptide profile
suggest that hrIDS is proteolitically processed ârandomlyâ
which agrees with the ultrafiltration assay in which the hrIDS activity
was found in all fractions (100kDa). No
glycation sites were found by computer analysis of the hIDS sequence;
discarding the possibility of marks for glycation and proteolytic
processing
Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris
Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an Xâlinked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronateâ2âsulfatase (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 oxygenâlimited conditions using a codonâoptimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mgâ1 Hâ1 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 doseâdependent 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