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Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications

Overview of attention for article published in ACS Chemical Biology, August 2017
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Title
Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications
Published in
ACS Chemical Biology, August 2017
DOI 10.1021/acschembio.7b00454
Pubmed ID
Authors

Shuchi Gaur, Aarohi Bhargava-Shah, Sharon Hori, Rayhaneh Afjei, Thillai V. Sekar, Sanjiv S. Gambhir, Tarik F. Massoud, Ramasamy Paulmurugan

Abstract

Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein it has increased sensitivity in vitro and in vivo (∼20,000-fold, and ∼1,000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention by both modifying the N-terminal secretory signal peptide, and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminal of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.

Twitter Demographics

The data shown below were collected from the profile of 1 tweeter who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 14 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 14 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 3 21%
Unspecified 2 14%
Professor 1 7%
Student > Bachelor 1 7%
Student > Doctoral Student 1 7%
Other 4 29%
Unknown 2 14%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 3 21%
Unspecified 2 14%
Agricultural and Biological Sciences 2 14%
Veterinary Science and Veterinary Medicine 1 7%
Environmental Science 1 7%
Other 2 14%
Unknown 3 21%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 03 August 2017.
All research outputs
#10,258,469
of 11,563,317 outputs
Outputs from ACS Chemical Biology
#1,679
of 1,837 outputs
Outputs of similar age
#224,213
of 265,371 outputs
Outputs of similar age from ACS Chemical Biology
#61
of 71 outputs
Altmetric has tracked 11,563,317 research outputs across all sources so far. This one is in the 1st percentile – i.e., 1% of other outputs scored the same or lower than it.
So far Altmetric has tracked 1,837 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 5.8. This one is in the 1st percentile – i.e., 1% of its peers scored the same or lower than it.
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We're also able to compare this research output to 71 others from the same source and published within six weeks on either side of this one. This one is in the 1st percentile – i.e., 1% of its contemporaries scored the same or lower than it.