Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum.
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.
Institut für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Federal Republic of Germany.
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Lemp
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Haselbeck
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Klebl
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004921:Erythropoietin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D015723:Gene Library; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000378:metabolism
D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004921:Erythropoietin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase