title: Highly efficient production of peptides: N-glycosidase F for N-glycomics analysis.
abstract: Peptide: N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacterium meningosepticum that serves as a useful tool in the research on protein N-glycosylation. However, native PNGase F purified from F. meningosepticum and recombinant PNGase F expressed in Escherichia coli are obtained only at low levels, with the culture yield being no more than 15mg/L. Here, we report the efficient production of large amounts of recombinant PNGase F. First, a codon-optimized sequence encoding F. meningosepticum PNGase F was cloned into the pPICZaA vector, which was used to transform Pichia pastoris GS115. Clones were screened directly by dot blotting with an anti-6His-tag antibody, and then protein expression was induced in glass tubes to conduct validation assays. The clone expressing the highest level of PNGase F was selected for fermentation at a 5-L scale, and then the recombinant enzyme produced was purified in a single step using affinity chromatography, which yielded 800mg of the protein per liter of culture. The partly glycosylated recombinant PNGase F exhibited an identical specific activity as commercially available PNGase F when using RNase B or other N-glycoproteins as substrates. Thus, the method developed in this study can facilitate the large-scale production and use of PNGase F in the rapidly developing research field of N-glycomics.
authors.affiliation: Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, Hubei, PR China; Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Ling
authors.initials: L
authors.lastname: Hua
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Xiaopan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Deyou
authors.initials: D
authors.lastname: Wan
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
authors.forename: Chunpeng
authors.initials: C
authors.lastname: He
authors.affiliation: Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan Street, Wuhan 430070, Hubei, PR China. Electronic address: caojiyue@mail.hzau.edu.cn.
authors.forename: Jiyue
authors.initials: J
authors.lastname: Cao
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, PR China. Electronic address: songhf259@163.com.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D054794:Glycomics; D006031:Glycosylation; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Deglycosylation activity; F. meningosepticum; N-Glycomics; Peptide: N-glycosidase F (PNGase F); Pichia pastoris