Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast.

Abstract:

: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.

journal_name

Protein Expr Purif

authors

Mrázek H,Benada O,Man P,Vaněk O,Křen V,Bezouška K,Weignerová L

doi

10.1016/j.pep.2011.09.009

subject

Has Abstract

pub_date

2012-01-01 00:00:00

pages

106-114

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(11)00266-X

journal_volume

81

pub_type

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