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.
Lemp
D
D
Institut für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Federal Republic of Germany.
Haselbeck
A
A
Klebl
F
F
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