Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum.
A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.
Tarentino
A L
AL
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Quinones
G
G
Trumble
A
A
Changchien
L M
LM
Duceman
B
B
Maley
F
F
Plummer
T H
TH
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase