Highly efficient production of peptides: N-glycosidase F for N-glycomics analysis
| Autor: | Xiaopan Yang, Chun-peng He, Wan Deyou, Xin Gao, Haifeng Song, Jiyue Cao, Ling Hua |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
PNGase F
Glycosylation RNase P Molecular Sequence Data Biology medicine.disease_cause Flavobacterium Pichia Amidase Pichia pastoris law.invention Bacterial Proteins Affinity chromatography law medicine Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Amino Acid Sequence Asparagine Cloning Molecular Glycomics Escherichia coli Base Sequence biology.organism_classification Molecular biology Recombinant Proteins Biochemistry Recombinant DNA Biotechnology |
| Zdroj: | Protein Expression and Purification. 97:17-22 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2014.02.004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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