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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

Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.
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pone.0120458.g006: Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.

Mentions: Endo H-P efficiently cleaved the N-glycan side-chains from RNase B at temperatures ranging from 30°C to 45°C, and 37°C was determined to be the optimum temperature (Fig. 6A). EndoH-P showed the highest activity at pH 5.5 to 6.5, and the optimum pH was 5.5 (Fig. 6B). These results were consistent with those obtained using commercial Endo H.


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)

Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120458.g006: Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.
Mentions: Endo H-P efficiently cleaved the N-glycan side-chains from RNase B at temperatures ranging from 30°C to 45°C, and 37°C was determined to be the optimum temperature (Fig. 6A). EndoH-P showed the highest activity at pH 5.5 to 6.5, and the optimum pH was 5.5 (Fig. 6B). These results were consistent with those obtained using commercial Endo H.

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