title: Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System.
abstract: The peptide-N (4)-(N-acetyl-β-D-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.
authors.affiliation: Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Masuda
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Takumi
authors.initials: T
authors.lastname: Mitsudome
authors.forename: Yudai
authors.initials: Y
authors.lastname: Nagata
authors.forename: Daisuke
authors.initials: D
authors.lastname: Morokuma
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mon
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Banno
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kusakabe
authors.forename: Jae Man
authors.initials: JM
authors.lastname: Lee
mesh_terms: D000818:Animals; D016367:Baculoviridae; D012831:Bombyx; D002460:Cell Line; D015870:Gene Expression; D019476:Insect Proteins / Q000096:biosynthesis* / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D019476:Insect Proteins; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
references.citation: J Biochem Biophys Methods. 1989;20(1):53-68
references.pmid: 2630586
references.citation: J Am Soc Mass Spectrom. 2000 Jun;11(6):564-71
references.pmid: 10833030
references.citation: J Biol Chem. 1988 Dec 5;263(34):18253-68
references.pmid: 2461366
references.citation: Proc Natl Acad Sci U S A. 1982 Aug;79(15):4540-4
references.pmid: 6812050
references.citation: Biochemistry. 1994 Oct 4;33(39):11699-706
references.pmid: 7918386
references.citation: Mol Biotechnol. 2008 Oct;40(2):180-5
references.pmid: 18543128
references.citation: Appl Biochem Biotechnol. 2014 Apr;172(8):3978-88
references.pmid: 24599668
references.citation: Protein Expr Purif. 2002 Feb;24(1):90-8
references.pmid: 11812228
references.citation: Biochim Biophys Acta. 1999 Dec 6;1473(1):4-8
references.pmid: 10580125
references.citation: Appl Microbiol Biotechnol. 2010 Jan;85(3):459-70
references.pmid: 19830419
references.citation: Biochem Biophys Res Commun. 2005 Jan 21;326(3):564-9
references.pmid: 15596136
references.citation: Appl Microbiol Biotechnol. 2013 Aug;97(15):6813-22
references.pmid: 23179626
references.citation: J Biol Chem. 1990 Sep 15;265(26):15606-10
references.pmid: 2203781
references.citation: Curr Opin Chem Biol. 1999 Dec;3(6):643-9
references.pmid: 10600722
references.citation: Carbohydr Res. 1994 Aug 17;261(2):173-86
references.pmid: 7954510
references.citation: Arch Biochem Biophys. 1995 Apr 20;318(2):465-75
references.pmid: 7733678
references.citation: Structure. 1994 Nov 15;2(11):1049-59
references.pmid: 7881905
references.citation: J Biol Chem. 1990 Apr 25;265(12):6967-72
references.pmid: 2182635
references.citation: J Biol Chem. 1986 Jan 5;261(1):172-7
references.pmid: 3941069
references.citation: Biotechnol Lett. 2012 Oct;34(10):1773-9
references.pmid: 22714272
references.citation: J Biol Chem. 1995 Dec 8;270(49):29493-7
references.pmid: 7493989
references.citation: J Biol Chem. 1990 Apr 25;265(12):6961-6
references.pmid: 2182634
references.citation: Eur J Biochem. 1991 Aug 1;199(3):647-52
references.pmid: 1868849
references.citation: J Biol Chem. 1984 Sep 10;259(17):10700-4
references.pmid: 6206060
references.citation: Protein Expr Purif. 2014 May;97:17-22
references.pmid: 24582822