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High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.

Wang F, Wang X, Yu X, Fu L, Fu L, Liu Y, Ma L, Zhai C - PLoS ONE (2015)

Bottom Line: The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris.Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation.This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

View Article: PubMed Central - PubMed

Affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China.

ABSTRACT
Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

No MeSH data available.


Related in: MedlinePlus

The hydrolytic activity of Endo H-P to proteins from mammals and baker's yeast.(A). The hydrolytic activity of Endo H-P to EPO expressed in CHO cells. Lane 1 EPO without treatment; Lane 2 EPO treated with commercial Endo H; Lane 3 EPO treated with purified Endo H-P; Lane 4 EPO treated with commercial PNGase F. (B). The hydrolytic activity of Endo H-P to CPY from baker's yeast. Lane 1 CPY without treatment; Lane 2 CPY treated with commercial Endo H; Lane 3 CPY treated with purified Endo H-P.
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pone.0120458.g005: The hydrolytic activity of Endo H-P to proteins from mammals and baker's yeast.(A). The hydrolytic activity of Endo H-P to EPO expressed in CHO cells. Lane 1 EPO without treatment; Lane 2 EPO treated with commercial Endo H; Lane 3 EPO treated with purified Endo H-P; Lane 4 EPO treated with commercial PNGase F. (B). The hydrolytic activity of Endo H-P to CPY from baker's yeast. Lane 1 CPY without treatment; Lane 2 CPY treated with commercial Endo H; Lane 3 CPY treated with purified Endo H-P.

Mentions: Meanwhile, the glycohydrolase activity of Endo H-P to other substrates from mammals and Baker's yeast was compared with recombinant Endo H from E. coli. Both of them could remove N-linked glycans of CPY and RNase B but not EPO, which was able to be cleaved by PNGase F (Fig. 5). This result indicated that Endo H-P expressed in different system was able to deglycosylate proteins from mammals, and the sensitivity was various to different glycoproteins.


High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.

Wang F, Wang X, Yu X, Fu L, Fu L, Liu Y, Ma L, Zhai C - PLoS ONE (2015)

The hydrolytic activity of Endo H-P to proteins from mammals and baker's yeast.(A). The hydrolytic activity of Endo H-P to EPO expressed in CHO cells. Lane 1 EPO without treatment; Lane 2 EPO treated with commercial Endo H; Lane 3 EPO treated with purified Endo H-P; Lane 4 EPO treated with commercial PNGase F. (B). The hydrolytic activity of Endo H-P to CPY from baker's yeast. Lane 1 CPY without treatment; Lane 2 CPY treated with commercial Endo H; Lane 3 CPY treated with purified Endo H-P.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362766&req=5

pone.0120458.g005: The hydrolytic activity of Endo H-P to proteins from mammals and baker's yeast.(A). The hydrolytic activity of Endo H-P to EPO expressed in CHO cells. Lane 1 EPO without treatment; Lane 2 EPO treated with commercial Endo H; Lane 3 EPO treated with purified Endo H-P; Lane 4 EPO treated with commercial PNGase F. (B). The hydrolytic activity of Endo H-P to CPY from baker's yeast. Lane 1 CPY without treatment; Lane 2 CPY treated with commercial Endo H; Lane 3 CPY treated with purified Endo H-P.
Mentions: Meanwhile, the glycohydrolase activity of Endo H-P to other substrates from mammals and Baker's yeast was compared with recombinant Endo H from E. coli. Both of them could remove N-linked glycans of CPY and RNase B but not EPO, which was able to be cleaved by PNGase F (Fig. 5). This result indicated that Endo H-P expressed in different system was able to deglycosylate proteins from mammals, and the sensitivity was various to different glycoproteins.

Bottom Line: The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris.Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation.This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

View Article: PubMed Central - PubMed

Affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China.

ABSTRACT
Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

No MeSH data available.


Related in: MedlinePlus