title: Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli.
abstract: The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.
authors.affiliation: Genzyme Corporation, Cambridge, Massachusetts 02142.
authors.forename: G D
authors.initials: GD
authors.lastname: Barsomian
authors.forename: T L
authors.initials: TL
authors.lastname: Johnson
authors.forename: M
authors.initials: M
authors.lastname: Borowski
authors.forename: J
authors.initials: J
authors.lastname: Denman
authors.forename: J F
authors.initials: JF
authors.lastname: Ollington
authors.forename: S
authors.initials: S
authors.lastname: Hirani
authors.forename: D S
authors.initials: DS
authors.lastname: McNeilly
authors.forename: J R
authors.initials: JR
authors.lastname: Rasmussen
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
chemical_list: D011994:Recombinant Proteins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase