Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens.
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.
H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark. snc@lundbeck.com
Søren
S
Christensen
Jan
J
Egebjerg
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004921:Erythropoietin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D005798:Genes, Bacterial; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D004921:Erythropoietin; D006639:Histidine; D009439:Neuraminidase