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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

Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
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pone.0120458.g004: Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.

Mentions: The fermentation supernatant was purified and the protein concentration was 61.9 mg/l after purification. Enzymatic activity was measured using denatured RNase B as the substrate. Endo H digestion results in the release of the glycan side-chains from RNase B, yielding a decrease in molecular weight from 17 kDa to approximately 15 kDa and a corresponding mobility shift during SDS-PAGE. As a control, 1U commercial Endo H (NEB, USA) was found to remove almost all of the carbohydrates from the denatured RNase B following incubation at 37°C for 1 h (Fig. 4). At least 1 μl of 3.5%-fold-diluted Endo H-P was required to achieve the same level of digestion (Fig. 4). Accordingly, the enzymatic activity of Endo H-P was determined to be approximately 461573 U/mg.


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)

Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120458.g004: Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B.M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
Mentions: The fermentation supernatant was purified and the protein concentration was 61.9 mg/l after purification. Enzymatic activity was measured using denatured RNase B as the substrate. Endo H digestion results in the release of the glycan side-chains from RNase B, yielding a decrease in molecular weight from 17 kDa to approximately 15 kDa and a corresponding mobility shift during SDS-PAGE. As a control, 1U commercial Endo H (NEB, USA) was found to remove almost all of the carbohydrates from the denatured RNase B following incubation at 37°C for 1 h (Fig. 4). At least 1 μl of 3.5%-fold-diluted Endo H-P was required to achieve the same level of digestion (Fig. 4). Accordingly, the enzymatic activity of Endo H-P was determined to be approximately 461573 U/mg.

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