The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide

Knappskog, Stian; Ravneberg, Hanne; Gjerdrum, Christine; Tröße, Christiane; Stern, Beate; Pryme, Ian F.

doi:10.1016/j.jbiotec.2006.11.026

Cited by 96 publications

(90 citation statements)

References 20 publications

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“…GLUC is a reporter enzyme from the marine copepod Gaussia princeps that is secreted from cells by signal-peptide mediated secretion. 47,48 Surprisingly, GLUC lacking the signal peptide rapidly exits the cell by a nonconventional pathway. 20 This pathway can be frustrated by anchoring an N-terminally deleted form of GLUC (dNGLUC) to the actin cytoskeleton.…”

Section: Quantitation Of Autophagymentioning

confidence: 99%

Quantitation of autophagy by luciferase release assay

Ketteler

Seed²

2008

Autophagy

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Autophagy is a cellular process that has been defined and analyzed almost entirely by qualitative measures. In no small part, this is attributable to the absence of robust quantitative assays that can easily and reliably permit the progress of key steps in autophagy to be assessed. We have recently developed a cell-based assay that specifically measures proteolytic cleavage of a tripartite sensor protein by the autophagy protease ATG4B. Activation of ATG4B results in release of Gaussia luciferase from cells that can be noninvasively harvested from cellular supernatants. Here, we compare this technique to existing methods and propose that this type of assay will be suitable for genome-wide functional screens and in vivo analysis of autophagy.

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Section: Quantitation Of Autophagymentioning

confidence: 99%

Quantitation of autophagy by luciferase release assay

Ketteler

Seed²

2008

Autophagy

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“…It has been reported that properties of residues at the h/c boundary and +1 position of mature protein can inXuence the translocation and cleavage of signal peptide (Nothwehr et al 1990;Barkocy-Gallagher and Bassford 1992). Besides that, the signal peptide can also aVect the regulation of gene expression (Serruto and Galeotti 2004), and it has an eVect on the secretion of protein production (Holden et al 2005;Knappskog et al 2007). After that, we compared the eYciency of two signal sequence from human and mouse nerve growth factor.…”

Section: Discussionmentioning

confidence: 99%

“…Our selected signal peptides belong to Type I. It is not only that the level of synthesis and secretion of proteins in cultured mammalian cells is heavily dependent on the choice of signal peptide (Knappskog et al 2007), but also that the optimization of signal peptides can increase the production of heterologous proteins (Ravn et al 2003). Apparently, our expressed betaendorphin would mature only if the signal peptide was cleaved oV from the "whole" protein, the signal peptide plus beta-endorphin.…”

Section: Discussionmentioning

confidence: 99%

Human signal peptide had advantage over mouse in secretory expression

Pei

Miao

et al. 2009

Histochem Cell Biol

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The signal peptide is a critical component in the secretory expression of protein in eukaryotic cells. It has been verified that the signal peptide of mouse nerve growth factor could mediate the secretory expression of beta-endorphin in cultured non-neuronal cells. Although there is a counterpart of nerve growth factor in human genome, no research about the signal sequence from human genome has been reported. The function of mediating secretory expression is affected by many factors. We assumed that the counterpart from human genome could function as the signal peptide from mouse nerve growth factor does and these two signal sequences had different efficiency in mediating secretory expression of beta-endorphin, but we could not figure out which one had a better function. To validate our hypothesis and give an answer to the question, we constructed two eukaryotic vectors, pcDNA3.1-hEP and pcDNA3.1-mEP, containing human and mouse signal sequences in fusion genes, respectively. RT-PCR showed that the constructed fusion genes were expressed in NIH3T3 cells. We also found that the detected beta-endorphin by the immunofluorescent technique was mainly in the cytoplasm of NIH3T3 cells. The concentration of beta-endorphin in the culture medium by RIA is 280.33 +/- 24.16 (pg/ml) and 191.04 +/- 7.96 (pg/ml) from pcDNA3.1-hEP and pcDNA3.1-mEP, respectively, and there was a significant statistical difference between them (P < 0.05). A difference existed between them and that from blank vector individually (P < 0.01). These findings suggest that our constructed fusion gene containing the signal sequence of human nerve growth factor can be secretorily expressed and the efficiency of the signal peptide from human nerve growth factor is higher than that of mouse signal peptide.

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“…To replace their signal peptide regions, the E1 and E2 genes minus the signal peptide were amplified by polymerase chain reaction (PCR) using long upstream primers. These primers were used to add the signal peptide of self-designed [21,22], tissue plasminogen activator (tPA) [23,24] or Gaussia luciferase (Gluc) [25] (Table 1), to the E1 and E2 genes. Each of these primers contained 15 nucleotides of the E1 or E2 sequence spanning the signal peptidase cleavage site.…”

Section: Methodsmentioning

confidence: 99%

Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins

Wen¹,

Deng²,

Gao³

et al. 2011

ABBS

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A large number of researches focused on glycoproteins E1 and E2 of hepatitis C virus (HCV) aimed at the development of anti-HCV vaccines and inhibitors. Enhancement of E1/E2 expression and secretion is critical for the characterization of these glycoproteins and thus for subunit vaccine development. In this study, we designed and synthesized three signal peptide sequences based on online programs SignalP, TargetP, and PSORT, then removed and replaced the signal peptide preceding E1/E2 by overlapping the polymerase chain reaction method. We assessed the effect of this alteration on E1/E2 expression and secretion in mammalian cells, using western blot analysis, dot blot, and Galanthus nivalis agglutinin lectin capture enzyme immunoassay. Replacing the peptides preceding E1 and E2 with the signal peptides of the tissue plasminogen activator and Gaussia luciferase resulted in maximum enhancement of E1/E2 expression and secretion of E1 in mammalian cells, without altering glycosylation. Such an advance would help to facilitate both the research of E1/E2 biology and the development of an effective HCV subunit vaccine. The strategy used in this study could be applied to the expression and production of other glycoproteins in mammalian cell line-based systems.

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The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide

Cited by 96 publications

References 20 publications

Quantitation of autophagy by luciferase release assay

Quantitation of autophagy by luciferase release assay

Human signal peptide had advantage over mouse in secretory expression

Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins

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