• ホーム
  • 検索結果
  • Production of a functional human acid maltase in tobacco seeds: biochemical analysis, uptake by human GSDII cells, and in vivo studies in GAA knockout mice.

Production of a functional human acid maltase in tobacco seeds: biochemical analysis, uptake by human GSDII cells, and in vivo studies in GAA knockout mice.

Applied biochemistry and biotechnology (2013-08-03)
Frank Martiniuk, Serena Reggi, Kam-Meng Tchou-Wong, William N Rom, Matteo Busconi, Corrado Fogher
要旨

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe's disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean β-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA(-/-)) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.

材料
製品番号
ブランド
製品内容

Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, lyophilized powder, 30-60 units/mg protein (biuret), ≤0.02% glucose
Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, lyophilized, powder, ~70 U/mg
Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, powder, white, ~120 U/mg
Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, ammonium sulfate suspension, ≥40 units/mg protein
Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, ≥260 U/mL, aqueous solution
Sigma-Aldrich
アミログルコシダーゼ from Aspergillus niger, Isoelectric focusing marker, pI 3.6
Sigma-Aldrich
アミログルコシダーゼ from Rhizopus sp., ≥40,000 units/g solid