FLAG-Frankenbody Expression Plasmids

Plasmids Encoding Anti-FLAG Single-Chain Variable Fragment (scFv) Fused with mEGFP or HaloTag Under CMV Promoter

At a Glance

Researchers at Colorado State University have developed a genetically encodable single chain variable fragment (scFv) that specifically binds the FLAG tag with high affinity in living cells (in vivo). This scFv can be used to quantify FLAG-tagged protein localization, translation, and dynamics in live cells.

Background

The FLAG tag (DYKDDDK) is one of the most commonly used and shortest linear epitope tags. It has been widely used for protein purification, immunoassays, and imaging. Because of its small size, the FLAG tag can be inserted into diverse locations of a protein of interest. This makes the FLAG tag ideal for labeling proteins that are too small or sensitive to larger tags, such as fluorescent fusion proteins.

Traditionally, the FLAG tag has been used for imaging purposes in immunofluorescence assays. While this makes it possible to image FLAG-tagged protein expression and localization in fixed (dead) cells, all dynamic information is lost. Furthermore, purified full-length anti-FLAG antibody is required, which can be costly. These issues have limited the experimental applications of the FLAG tag.

Overview

Aimed at enabling research in live cells and organisms, the Stasevich lab has developed a genetically encodable single chain variable fragment (scFv) that binds the FLAG tag in live systems, named the anti-FLAG frankenbody. The engineered anti-FLAG frankenbody is based on an anti-FLAG antibody sequence supplied by the FUJIFILM Wako Pure Chemical Corporation.

That antibody binds the FLAG tag specifically and tightly, but the binding capability is lost when the antibody is converted to a genetically encodable scFv format and expressed in live systems. This is due to interruption of scFv folding and disulfide-bond formation in the reduced environment of live cells.

To address this issue, the original anti-FLAG scFv complementary determining regions were grafted onto a more stable scFv scaffold that had high sequence similarity with the original scFv. After loop grafting, the resulting two anti-FLAG frankenbodies were shown to bind the FLAG tag specifically and tightly in live systems. When either of these anti-FLAG frankenbodies are fused to fluorescent proteins and co-expressed in live cells/organisms harboring FLAG-tagged proteins, the FLAG-tagged proteins can be tracked in real time.

A unique application of anti-FLAG frankenbodies for live-cell imaging is the ability to monitor single-mRNA translation kinetics. This works because anti-FLAG frankenbodies can bind FLAG tags very rapidly, so newly-translated FLAG tags are bound and labeled even before synthesis of the full-length FLAG-tagged protein is complete. This is not possible using more traditional fluorescent fusion tags like GFP because these traditional tags take too long to mature and fluoresce.

Anti-FLAG frankenbodies can be fused to different colored fluorescent proteins to image FLAG-tagged proteins in a rainbow of colors.

Flag-Frankenbody Expression Plasmids

Kerafast FLAG- Frankenbody GFP or HALO info/purchase

Addgene FLAG- Frankenbody GFP or HALO info/purchase

Benefits

Anti-FLAG frankenbodies can be fused to different colored fluorescent proteins to image FLAG-tagged proteins in a rainbow of colors.

  • Genetically encodable anti-FLAG frankenbody can be expressed or produced in live cells
  • The small size of the FLAG tag limits interference with underlying biology
  • The FLAG tag (DYKDDDDK) is common in biotechnology
  • The ability to image the dynamics of FLAG-tagged proteins in live cells and organisms

Applications

  • Live cell imaging
  • Imaging of FLAG-tagged protein translation dynamics with single-mRNA resolution
  • RNA-protein interactions
Last Updated: March 2023
The plasmid being used in live cell imaging
Opportunity

Available for Purchase at Kerafast and Addgene

Inventors

Timothy Stasevich
Ning Zhao
Hiroshi Kimura
Yuko Sato

Reference Number
2020-009
Licensing Manager

Steve Foster
Steve.Foster@colostate.edu
970-491-7100