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Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants.

ABSTRACT: Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on protein production and provide evidence that N-glycosylation greatly increases the expression levels of ER-targeted recombinant proteins. Fusion of the extracellular domain (M domain) of protein tyrosine phosphatase receptor type C (CD45), which contains four putative N-glycosylation sites to a model protein, leptin at the C-terminus, increased recombinant protein levels by 6.1 fold. This increase was specific to ER-targeted proteins and was dependent on N-glycosylation. Moreover, expression levels of leptin, leukemia inhibitory factor and GFP were also greatly increased by fusion of M domain at either the N or C-terminus. Furthermore, the increase in protein levels resulted from enhanced translation, but not transcription. Based on these results, we propose that fusing a small domain containing N-glycosylation sites to target proteins is a powerful technique for increasing the expression levels of recombinant proteins in plants.

SUBMITTER: Kang H

PROVIDER: S-EPMC5854594 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants.

Kang Hyangju H Park Youngmin Y Lee Yongjik Y Yoo Yun-Joo YJ Hwang Inhwan I

Scientific reports 20180315 1

Plants represent promising systems for producing various recombinant proteins. One key area of focus for improving this technology is developing methods for producing recombinant proteins at high levels. Many methods have been developed to increase the transcript levels of recombinant genes. However, methods for increasing protein production involving steps downstream of transcription, including translation, have not been fully explored. Here, we investigated the effects of N-glycosylation on pr ...[more]

PMID: 29545574

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Project description:Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.

Dataset Information

Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants.

Publications

Fusion of a highly N-glycosylated polypeptide increases the expression of ER-localized proteins in plants.

Similar Datasets

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