Highly efficient production of peptides: N-glycosidase F for N-glycomics analysis

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• 作者：Ling Hua^a ; ^b ; Xin Gao^b ; Xiaopan Yang^b ; Deyou Wan^b ; Chunpeng He^b ; Jiyue Cao^a ; ^{caojiyue@mail.hzau.edu.cn" class="auth_mail} ; Haifeng Song^b ; ^{songhf259@163.com" class="auth_mail}
• 关键词：Peptide ; N-glycosidase F (PNGase F) ; F. meningosepticum ; Pichia pastoris ; N-Glycomics ; Deglycosylation activity
• 刊名：Protein Expression and Purification
• 出版年：May, 2014
• 年：2014
• 卷：97
• 期：Complete
• 页码：17-22
• 全文大小：1441 K

文摘

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.