Pimentel Natalia, Rodríguez-Lopez Alexander, Díaz Sergio, Losada Juan C, Díaz-Rincón Dennis J, Cardona Carolina, Espejo-Mojica Ángela J, Ramírez Aura M, Ruiz Fredy, Landázuri Patricia, Poutou-Piñales Raúl A, Cordoba-Ruiz Henry A, Alméciga-Díaz Carlos J, Barrera-Avellaneda Luis A
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, Colombia.
Chemical Department, Faculty of Science, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
Biotechnol Appl Biochem. 2018 Sep;65(5):655-664. doi: 10.1002/bab.1660. Epub 2018 Apr 23.
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.
亨特综合征（黏多糖贮积症II型，MPS II）是一种X连锁溶酶体贮积病，由溶酶体酶艾杜糖醛酸-2-硫酸酯酶（IDS）缺乏所致。目前，MPS II患者主要接受酶替代疗法（ERT），使用哺乳动物细胞生产的重组酶。作为一种替代方法，多项研究表明可在微生物中生产有活性和治疗作用的溶酶体蛋白。在本文中，我们报告了在毕赤酵母中生产的重组IDS（prIDS）的制备及特性。我们评估了培养条件和基因序列优化对prIDS生产的影响。结果表明，使用密码子优化的IDS cDNA，在限氧条件下可获得最高产量的prIDS。纯化后的酶最终活性为12.45 nmol mg H，表观分子量约为90 kDa。在pH 6.0时稳定性最高，prIDS在人血清中也表现出高稳定性。值得注意的是，该酶通过甘露糖受体以剂量依赖的方式被培养细胞摄取，从而使酶能够递送至溶酶体。总之，这些结果表明毕赤酵母作为生产用于MPS II ERT的IDS的宿主具有潜力。
