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.
Highlights
System [24], and previous reports have indicated that over-glycosylation may enhance the thermodynamic and kinetic stability of recombinant proteins [25], some studies have indicated that the hyper-glycosylation that occurs in P. pastoris may compromise function [26,27]
Glycohydrolases are important for identifying the composition of the glycan portion of glycoproteins but are useful for preparing active enzymes Endo H is important among glycohydrolases
The recombinant Endo H from P. pastoris and E. coli could remove N-linked glycan from mammal and Baker's yeast glycoproteins, such as RNase B and Carboxypeptidase Y from baker's yeast (CPY). It could cleave polysaccharides from glycoproteins heterolgously expressed in P. pastoris
Summary
We attempted to use recombinant Endo H to deglycosylate the recombinant proteins expressed and modified by P. pastoris through both co-fermentation and post-fermentation treatments. Recombinant P. pastoris strains expressing endo-1, 4-β-mannosidase from Aspergillus niger, DNase I from Bos taurus, phytase from E. coli and DpnI from Diplococcus pneumoniae were For the post-fermentation treatment, the recombinant P. pastoris strains producing endo-1, 4β-mannosidase, phytase and DpnI were incubated in 100 ml BMGY until the OD600 value reached approximately 20.
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