Newsletter: Fluorescent Protein Expression

Fluorescent proteins are an important part of any molecular biologist’s toolkit. Recombinant DNA techniques allow researchers to incorporate a fluorescent protein into an engineered plasmid and track its expression over time with flow cytometry or, more commonly, fluorescent microscopy methods. Here we explore the history, advantages, common uses and wide selection of fluorescent proteins.

History

The bioluminescent proteins green fluorescent protein (GFP) and aequorin were first isolated from the jellyfish species Aequorea Victoria in 1961 (PMID: 13911999). However, this discovery remained fairly unknown until the gene coding for GFP was sequenced and cloned in 1992 (PMID: 1347277). Subsequent papers demonstrated the many potential research applications for GFP (PMID: 8682203), leading to the widespread use of recombinant fluorescent probes to observe a wide variety of cellular processes in living systems.

Advantages

Fluorescent proteins offer several advantages over alternative fluorescent molecules.

• Fluorescent proteins do not require fixation, enzymes, or substrates to generate fluorescence.
• While FITC is highly phototoxic, illuminating GFP is generally not harmful to living cells. This allows researchers to observe cells over time, without affecting their long-term viability.
• When introduced properly, fluorescent-tagged proteins can be continually expressed and even passed on to subsequent generations of cells, allowing for longer-term studies of protein expression in cells or tissue. This heritability has even led to the creation of countless stable GFP-expressing cell lines and transgenic animal models.
• Fluorescent protein expression can be specifically controlled via inducible promoters. This allows researchers to activate GFP expression at the exact time required for their experiment.

Applications

Fluorescent proteins are most often used in reporter assays to identify if/when a specific gene is being expressed by a cell, tissue or organism. A few common examples include:

• As transformational reporters. Inserting a fluorescent protein gene into the same DNA construct being introduced to cells allows researchers to visually confirm that their transformation was successful. FACS analysis can then be used to enrich populations for positively transformed cells.
• As direct transcriptional and translational reporters
• As quantitative reporters of gene expression (PMID: 15640280)

They are also useful for mapping gene expression during cellular interactions or tissue development (PMID: 14671301)

Colors

Random and directed mutagenesis efforts on the wild type GFP have generated new genetic variants with enhanced brightness (EGFP) (PMID: 8707053) and folding characteristics (sfGFP) (PMID: 16369541), as well as many spectral variants ranging from yellow (YFP) (PMID: 11753368) to blue (EBFP) and cyan (ECFP).

Additional naturally occurring fluorescent proteins have been identified in other marine species from the phylum Cnidaria, including reef coral Anthozoa species (PMID: 10504696) and sea anemone Discosoma species (PMID: 23000031). These include red (dsRed) and orange variants which have subsequently undergone mutagenesis efforts to improve folding and spectral characteristics.

To date, a broad range of fluorescent proteins have been discovered or developed to span the visible light spectrum (PMID: 19771335). This table details the a few of the most common fluorescent protein options available on FluoroFinder’s experiment design tool.

Fluorescent proteins are a powerful tool for researchers monitoring gene expression or performing any molecular cloning experiments. These proteins offer many unique advantages over alternative fluorescent or colorimetric reporter assays, and the discovery and development of new fluorescent proteins have further expanded this important toolkit.