Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

Sagt, Cees M. J.; Kleizen, Bertrand; Verwaal, René; Jong, Margo D.M. de; Müller, Wally H.; Smits, Allan W.; Visser, Chris J.; Boonstra, Johannes; Verkleij, Arie J.; Verrips, C. Theo

doi:10.1128/aem.66.11.4940-4944.2000

Cited by 82 publications

(56 citation statements)

References 24 publications

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“…16 The C-terminal N-glycosylation has been generally considered not to be effectively occupied or functional because the terminated polypeptide chain can be dropped out from the translational complex before it can reach the catalytic site of OST and because the polypeptide chain has been already folded (or misfolded) before the glycan can be attached. 17,18 However, five of the six N- Cells were cultured and transfected as indicated in the Experimental section. Time of culture means the time of cell growth post-transfection.…”

Section: Discussionmentioning

confidence: 99%

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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N-glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N-glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N-glycosylation sequons, were tested by this method. Our results indicate that N-glycosylation sequons located at the N-or C-terminal are glycosylated at high rates and thus the N-and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N-glycosylation, presumably by improving their folding in the endoplasmic reticulum. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1468Prog., 25: -1475Prog., 25: , 2009 Keywords: N-glycosylation, protein expression, secretion, protein folding, mutagenesis, quality control IntroductionAsparagine glycosylation (N-glycosylation) is an important process for the delivery of secretory and membrane proteins to the extracellular space or cell surface. Thus, most secretory and membrane proteins contain asparagine-linked (N-linked) glycans, with one or more oligosaccharide chains attached to the polypeptide backbone. For some proteins, Nglycosylation is absolutely required for their secretion. For others, it may not be necessary. Nevertheless, oligosaccharide chains have been shown to be the important parts of the proteins and may play roles in the folding, transport, degradation, structure, stability, receptor-recognition, cellular localization, or other biological activities of the glycoproteins (reviewed in Refs. 1-3). Protein N-glycosylation takes place at the membrane of the endoplasmic reticulum (ER). The ER plays a primary quality control function to ensure the fidelity and proper folding of proteins before they travel out of the compartment, as incorrectly folded proteins can be toxic to the cell. A presynthesized oligosaccharide chain on a lipid carrier, dolichol, is transferred onto the asparagine residue in the NXS/T sequon of the nascent polypeptide by the oligosaccharyltransferase (OST) complex while translation is in process. 2 The attached oligosaccharide chains continue to be modified along the maturation pathway of the proteins in the ER and the N-glycosylation status will dictate the fate of the synthesized protein. The newly synthesized protein can proceed to secretion, aggregation, or degradation depending on the status and pattern of its N-glycosylation. [4][5][6] In the overexpression and production of recombinant proteins, the efficiency of N-glycosylation will affect the yield of the protein recovery and the circulation life of a pharmaceutical molecule. Although N-glycosylation is important, it is well known that not all potential glycosylation sites are attached with oligosaccharide chains. In our routine expression experi...

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Section: Discussionmentioning

confidence: 99%

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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“…Lastly, the hyperglycosylated linker could have a positive role in secretion, increasing the production yield as demonstrated for the hyperglycosylated linker from the A. niger glucoamylase (32). Indeed, glycosylation sites due to the presence of one or two linkers for FLX and FLXLC, respectively, could extend retention of recombinant proteins in the secretion pathway, thereby providing additional time for correct processing and resulting in an increase of production (41). This latter hypothesis could explain why the production yields for both bifunctional enzymes were higher than those obtained for the corresponding free recombinant enzymes (29,40).…”

Section: Discussionmentioning

confidence: 99%

Construction of Engineered Bifunctional Enzymes and Their Overproduction in Aspergillus niger for Improved Enzymatic Tools To Degrade Agricultural By-Products

Levasseur¹,

Navarro²,

Punt

et al. 2005

Appl Environ Microbiol

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Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a K d of 9.9 ؋ 10 ؊8 M for the dissociation constant and 0.98 mol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.

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“…Approaches like the use of appropriate promoters and their modified versions (26) and the use of fusions of signal peptides to the heterologous proteins for efficient targeting to the secretory pathway (1) have been used successfully for production of some proteins (reviewed in reference 25). Also, manipulation of glycosylation of the foreign protein has been shown to increase secretion (29). There are also some cases in which overexpression of genes of the secretory pathway has been shown to increase protein secretion.…”

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confidence: 99%

Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae

Valkonen

Penttilä

Saloheimo

2003

Appl Environ Microbiol

168

104

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One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens ␣-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both ␣-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in ␣-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of ␣-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.

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Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

Cited by 82 publications

References 24 publications

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Construction of Engineered Bifunctional Enzymes and Their Overproduction in Aspergillus niger for Improved Enzymatic Tools To Degrade Agricultural By-Products

Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae

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