Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.
We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.
Liu
J
J
Center for Molecular Biology and Gene Therapy and Department of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350, USA.
Wang
Y
Y
Szalay
A A
AA
Escher
A
A
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D003063:Cnidaria; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D007376:Interleukin-2 / Q000032:analysis* / Q000235:genetics; D008156:Luciferases / Q000032:analysis*; D049449:Luminescence; D008164:Luminescent Proteins / Q000032:analysis*; D021382:Protein Sorting Signals / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D037401:Scyphozoa; D014162:Transfection
D007376:Interleukin-2; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases