Abstract
N-Glycosidase F (peptide-N4-(N-acetyl-beta-glycosaminyl)asparagine amidase; EC 3.5.1.52) catalyzes the cleavage of N-glycosidically linked carbohydrate chains between N-acetylglucosamine and asparagine. The structural gene was isolated by screening a Flavobacterium meningosepticum genomic DNA library in lambda gt10 with oligonucleotides, deduced from partial amino acid sequences of the protein. A clone with an open reading frame of 1062 bases was obtained. The amino acid sequence reveals a 42-residue-long leader peptide, which shows similarities to the endoglycosidase H-leader with respect to the cleavage site of the signal peptide, but is distinct from the ones known from other Gram-positive or -negative bacteria. The molecular weight of the native protein, derived from the DNA sequence, is in agreement with the molecular weight of the purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35,000). Escherichia coli, transformed with a plasmid containing this DNA sequence, expresses N-glycosidase F activity. The enzyme with its natural Flavobacterium promoter and leader peptide is not secreted in E. coli but seems to be associated with cell membranes.
Highlights
From the Slnstitut fiir Zellbiologie und Pflanzenphysiologie, Universitiit Regensburg, Germany and §Biochemica Research Center, Boehringer Mannheim GmbH, D-8132
Endo H since it acts as an amidase by cleaving the /3
Structural differences in the leader peptide might be responsible for this lacking ability of E. coli to secrete the enzyme, albeit both leader sequences have exactly the same length (42 amino acids)
Summary
Meningosepticum (ATCC 33958) was used for preparing genomic DNA. The following Escherichia coli strains were used: DH5n IF-, endAl, hsdR17(rk,mk), supE44, thi-1, recA1, gyrA96, relA1, 080&acZAM15], C600 [F-, thi-1, thr-1, leuB6, lacY1, tonA21. E. coli, LB, supplemented with ampicillin (100 fig/ml) and 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (40 pg/ml) and isopropylthiogalactoside (20 mM), when required. For growing strains C600 and C600hfl, 10 mM MgCl, and 0.2% maltose was included in LB. X-DNA was prepared from plate-lysates, where agarose was used instead of agar [11]. No 903 299); erythropoietin, neuraminidase (Arthrobacter ureafaciens), glycan detection kit, PNA (digoxigenin-labeled), N-glycopeptide (/3-galactosidase-labeled), 2-nitrophenyl$-D-galactopyranoside, isopropylthiogalactoside, and 5-bromo-4-chloro-3-indolyl$-D-galactopyranosid (X-Gal) were from Boehringer Mannheim. Inc. and all other chemicals were of the highest quality available
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