Abstract
A new system based on non-conventional secretion of the luciferase from Gaussia princeps (GLUC) can be used to detect intracellular proteolysis in vivo.
Highlights
Advances in automation and the availability of genomic sequence information have led to the development of sophisticated cell-based assays for high-throughput screening of functional phenotypes [1]
By contrast, when dNGLUC was fused to the carboxyl terminus of β-actin, less than 1.5% of G. princeps luciferase (GLUC) activity was detected in SN (Table 1), and the relative light unit values observed were close to background
Cells expressing dNGLUC were exposed to 7 μM Monensin, 10 μg/ml Brefeldin A or 5 μg/ml MG132 and the activity accumulating over 4 h at 37°C was determined (Figure 1a)
Summary
Advances in automation and the availability of genomic sequence information have led to the development of sophisticated cell-based assays for high-throughput screening of functional phenotypes [1]. Most cell-based assays rely on fluorescent or luminescent reporters such as green fluorescent protein (GFP), secreted alkaline phosphatase (SEAP) or Photinus luciferase. Secreted luciferases offer many advantages over cellular reporter enzymes as they can be nondestructively harvested from cellular supernatants over time. Several secreted luciferases have been reported, from the marine copepods Gaussia princeps [2], and Metridia longa [3], the ostracod Vargula hilgendorfii [4], the decapod shrimp Oplophorus gracilirostris [5] and the ostracod crustacean Cypridina noctiluca [6]. Intracellular luciferases, such as from the sea pansy Renilla reniformis, can be engineered to be secreted and stable in the extra-cellular milieu [7]. A cDNA encoding G. princeps luciferase (GLUC) activity has recently been isolated and found to direct the synthesis of a
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