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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

Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from P. pastoris bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of P. pastoris expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of P. pastoris expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.
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pone.0120458.g011: Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from P. pastoris bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of P. pastoris expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of P. pastoris expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.

Mentions: No DNA band can be detected after pHBM905A plasmid was treated with DNase I of the glycosylated form or deglycosylated form obtained from co- or post-fermentation(Fig. 11 A). Meanwhile, the konjac powder in the plate was degraded by the endo-1, 4-β-mannosidase with the same treatment and a clear halo could be detected around the samples (Fig. 11 B). These results indicated that both enzymes still remained active after deglycosylation.


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)

Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from P. pastoris bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of P. pastoris expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of P. pastoris expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362766&req=5

pone.0120458.g011: Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from P. pastoris bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of P. pastoris expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of P. pastoris expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.
Mentions: No DNA band can be detected after pHBM905A plasmid was treated with DNase I of the glycosylated form or deglycosylated form obtained from co- or post-fermentation(Fig. 11 A). Meanwhile, the konjac powder in the plate was degraded by the endo-1, 4-β-mannosidase with the same treatment and a clear halo could be detected around the samples (Fig. 11 B). These results indicated that both enzymes still remained active after deglycosylation.

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