期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 97, 期 -, 页码 17-22出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2014.02.004
关键词
Peptide: N-glycosidase F (PNGase F); F. meningosepticum; Pichia pastoris; N-Glycomics; Deglycosylation activity
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 15 mg/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 800 mg 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. (C) 2014 Published by Elsevier Inc.
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