Dataset Information

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

ABSTRACT: Methylotrophic yeasts have widely been used as model organisms for understanding cellular functions and biochemical activities in lower eukaryotes. The gene encoding an aspartic protease (MCAP) from Mucor circinelloides DSM 2183 was cloned and expressed into Pichia pastoris using both the native M. circinelloides signal peptide (mcSP) and ?-factor secretion signal from Saccharomyces cerevisiae (?-MF). When expressed in P. pastoris using ?-MF and mcSP, MCAP was secreted into the culture medium at a concentration 200 mg L^-1 (410 MCU mL^-1) and 110 mg L^-1 (249 MCU mL^-1), respectively. The SDS-PAGE analysis of each culture shows that the protein was secreted in the media in two forms with molecular weights of approximately 33 and 37 kDa. Upon digestion using endoglycosidase H (Endo H), only one band at 33 kDa was observed, indicating that the protein might be glycosylated. One putative N-glycosylation site was found and a site-directed mutagenesis at position Asn331-Gln of the sequence produce only one form of the protein of 33 kDa, similar to that obtained when digested with Endo H. The optimum temperature and pH activity of the expressed MCAP was found to be at 60 °C and 3.6, respectively.

SUBMITTER: Kangwa M

PROVIDER: S-EPMC6167268 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

Kangwa Martin M Salgado Jose Antonio Gama JAG Fernandez-Lahore Hector Marcelo HM

AMB Express 20181001 1

Methylotrophic yeasts have widely been used as model organisms for understanding cellular functions and biochemical activities in lower eukaryotes. The gene encoding an aspartic protease (MCAP) from Mucor circinelloides DSM 2183 was cloned and expressed into Pichia pastoris using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF). When expressed in P. pastoris using α-MF and mcSP, MCAP was secreted into the culture medium at ...[more]

PMID: 30276572

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Project description:BackgroundN-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion.ResultsAdding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin.ConclusionsIn conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris.

Dataset Information

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

Publications

Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability.

Similar Datasets

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