Mrázek Hynek, Benada Oldřich, Man Petr, Vaněk Ondřej, Křen Vladimír, Bezouška Karel, Weignerová Lenka
Laboratory of Protein Architecture, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague, Czech Republic.
Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
Protein Expr Purif. 2012 Jan;81(1):106-114. doi: 10.1016/j.pep.2011.09.009. Epub 2011 Sep 29.
α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.
α-N-乙酰半乳糖胺酶（α-GalNAc-ase；EC.3.2.1.49）是一种外切糖苷酶，特异性水解各种糖链中末端α-连接的N-乙酰半乳糖胺。从黑曲霉中克隆了与α-GalNAc-ase基因对应的cDNA，进行测序，并在酿酒酵母中表达。α-GalNAc-ase基因包含一个开放阅读框，编码一个由487个氨基酸残基组成的蛋白质。从该阅读框的氨基酸序列推导的成熟蛋白分子量为54 kDa。重组蛋白被纯化至表观均一，并进行了生化特性鉴定（pI 4.4，对2-硝基苯基2-乙酰氨基-2-脱氧-α-D-吡喃半乳糖苷的K(M)为0.56 mmol/l，在pH2.0-2.4和50-55°C时达到最佳酶活性）。通过分析超速离心测量和动态光散射测定其分子量。我们的实验证实，与纯单体的天然形式相比，重组α-GalNAc-ase以两种不同的形式（70和130 kDa）存在。在野生型和重组α-GalNAc-ase的八个潜在N-糖基化位点中的六个位点证实了N-糖基化。
