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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.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.

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

Co-fermentaion of EndoH-P with the recombiant DNase I and phytase.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in P. pastoris; Lane 2 phytase co-induced with Endo H-P in P. pastoris; Lane 3 DNase I expressed in P. pastoris; Lane 4 DNase I co-induced with Endo H-P in P. pastoris; Lane 5 Endo H-P expressed in P. pastoris.
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pone.0120458.g008: Co-fermentaion of EndoH-P with the recombiant DNase I and phytase.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in P. pastoris; Lane 2 phytase co-induced with Endo H-P in P. pastoris; Lane 3 DNase I expressed in P. pastoris; Lane 4 DNase I co-induced with Endo H-P in P. pastoris; Lane 5 Endo H-P expressed in P. pastoris.

Mentions: The predicted sizes of E. coli phytase and Bos Taurus DNase I were 47 kDa and 31 kDa, respectively, whereas upon expression in P. pastoris, the observedsizes of these two proteins were much bigger due to glycosylation (Fig. 8, open arrows). The apparent sizesdecreased to match the predicted sizes after co-fermentation with the Endo H-P-expressing strain in shake flasks (Fig. 8, black arrows). Moreover, two main bands in close proximity were detected for the recombiant phytase, and only one smaller band was observed in the corresponding co-fermentation sample. These results were consistent with the post-fermenation treatment, indicating that both the post-fermentation and co-fermentaion treatments remove glycan chains in the same manner.


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)

Co-fermentaion of EndoH-P with the recombiant DNase I and phytase.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in P. pastoris; Lane 2 phytase co-induced with Endo H-P in P. pastoris; Lane 3 DNase I expressed in P. pastoris; Lane 4 DNase I co-induced with Endo H-P in P. pastoris; Lane 5 Endo H-P expressed in P. pastoris.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120458.g008: Co-fermentaion of EndoH-P with the recombiant DNase I and phytase.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 phytase expressed in P. pastoris; Lane 2 phytase co-induced with Endo H-P in P. pastoris; Lane 3 DNase I expressed in P. pastoris; Lane 4 DNase I co-induced with Endo H-P in P. pastoris; Lane 5 Endo H-P expressed in P. pastoris.
Mentions: The predicted sizes of E. coli phytase and Bos Taurus DNase I were 47 kDa and 31 kDa, respectively, whereas upon expression in P. pastoris, the observedsizes of these two proteins were much bigger due to glycosylation (Fig. 8, open arrows). The apparent sizesdecreased to match the predicted sizes after co-fermentation with the Endo H-P-expressing strain in shake flasks (Fig. 8, black arrows). Moreover, two main bands in close proximity were detected for the recombiant phytase, and only one smaller band was observed in the corresponding co-fermentation sample. These results were consistent with the post-fermenation treatment, indicating that both the post-fermentation and co-fermentaion treatments remove glycan chains in the same manner.

Bottom Line: The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris.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.

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