Article ; Online: A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.
2020 Volume 10, Issue 1, Page(s) 8953
Abstract: The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately ... ...
| Abstract | The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. |
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| MeSH term(s) | CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Luminescent Measurements/methods ; Luminescent Proteins/analysis ; Plasmids ; Proteins/analysis |
| Chemical Substances | Luminescent Proteins ; Proteins |
| Language | English |
| Publishing date | 2020-06-02 |
| Publishing country | England |
| Document type | Journal Article |
| ZDB-ID | 2615211-3 |
| ISSN | 2045-2322 ; 2045-2322 |
| ISSN (online) | 2045-2322 |
| ISSN | 2045-2322 |
| DOI | 10.1038/s41598-020-65832-1 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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