Abstract
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.
Highlights
N4-(N-acetyl-&glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli
The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Fluuobacterium peptide-N4-(N-acetyl-fi-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides
Peptide-N4-(N-acetyl-fi-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
Summary
’ Residue numbers represent the amino acid sequence determined by cDNA analysis. ’ Number designations of peptides from limited tryptic (LT) digestion of S-carboxymethyl-PNGase. ’ Residue numbers represent the amino acid sequence determined by cDNA analysis. ’ Number designations of peptides from limited tryptic (LT) digestion of S-carboxymethyl-PNGase. E Sequence aided by PTH degradation of smaller peptide fragments produced by chymotryptic digestion. F Sequence aided by PTH degradation of smaller peptide fragments produced by tryptic digestion. A Number designations of peptides isolated from tryptic (T) digestion of S-carboxymethyl-PNGase. H Number designations of peptides isolated from cyanogen bromide (CB) cleavage of PNGase F
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