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Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens
Author(s) -
Christensen Søren,
Egebjerg Jan
Publication year - 2005
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20040144
Subject(s) - sialidase , open reading frame , cloning (programming) , escherichia coli , biology , neuraminidase , gene , biochemistry , molecular cloning , expression vector , microbiology and biotechnology , peptide sequence , recombinant dna , enzyme , computer science , programming language
Sialidases have recently been used in the processing of clinically relevant asialoproteins. The Arthrobacter ureafaciens sialidase (EC 3.2.1.18) exhibits broad substrate specificity and is often used in such applications. We have employed an expression cloning strategy to isolate the A. ureafaciens sialidase. The clone encodes a 990‐amino‐acid 104 kDa open‐reading‐frame protein containing three domains: an N‐terminal catalytic domain, a linker domain with an immunoglobulin‐like fold and a C‐terminal domain of unknown function. Expression in Escherichia coli indicates that the sialidase promoter was active in E. coli . Overexpression in E. coli resulted in several truncated forms. A 54 kDa truncated variant was generated, expressed and purified, and its feasibility for use in an erythropoietin desialylation process was demonstrated.