BioChem - DOS

High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins

PloS one

Wang, Fei; Wang, Xiaojuan; Yu, Xiaolan; Fu, Lin; Liu, Yunyun; Ma, Lixin; Zhai, Chao

<P>Endo-&beta;-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-&beta;-N-acetylglucosaminidase H expression in <I>Pichia pastoris</I>. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of <I>Pichia pastoris</I> 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 <I>Escherichia coli</I> 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 <I>Pichia pastoris</I> 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-&beta;-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-&beta;-N-acetylglucosaminidase H expression in <I>Pichia pastoris</I> and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in <I>Pichia pastoris</I> using simplified methods.</P>

2015

Public Library of Science

cc-by

1932-6203

10

3

pp.e0120458

10.1371/journal.pone.0120458

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART73948710

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0120458&type=printable