KEY TECHNIQUE
Protein Localization Using Fluorescent
Fusion Proteins
PROBLEM: Techniques such as immunostaining can local-
ize proteins of interest in cells or whole organisms. But how can
the location of a specific protein be monitored in a living cell?
SOLUTION: DNA encoding a protein of interest can be
engineered so that the resulting protein is “tagged” with a fluo-
rescent protein. The resulting DNA can be introduced into cells,
which produce the tagged protein. Using fluorescence micros-
copy, the location of the fluorescent protein of interest can then
be followed in living cells.
Key Tools: Fluorescence microscopy (see Appendix, Figure
A-12); a known DNA sequence that encodes a protein of inter-
est; standard techniques for cloning and expressing DNA (see
Key Technique in Chapter 21, pages 648–649).
Bacteria Expressing Green Fluorescent Protein Variants.
Details: In this and previous chapters you learned that the
DNA sequence of the coding region of a gene indirectly pro-
vides the information cells need to produce a protein. When a
DNA sequence with the appropriate promoter is transcribed, if
the resulting RNA has the appropriate features, it can be trans-
lated into a protein. Scientists can capitalize on this ­relationship
Other common processing events include chemical modi-
fications of individual amino acid groups—by methylation,
phosphorylation, or acetylation reactions, for example. In ad-
dition, a polypeptide may undergo glycosylation (the addition
of carbohydrate side chains; see Chapter 12) or may be associ-
ated with a prosthetic group. Hemoglobin is a good example
of the latter: each of its four globin subunits contains a heme
group (Figure 3-4). Finally, in the case of proteins composed
of multiple subunits, individual polypeptide chains must bind
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between DNA and protein sequences in the laboratory to produce
fusion proteins. By fusing DNA sequences encoding a protein of
interest with DNA encoding a portion of another protein that fluo-
resces, scientists can produce DNA that encodes a fluorescently
tagged protein. When introduced into cells or an organism, the
protein produced from this DNA can be visualized in living cells.
Other types of fluorescent constructs are useful for studying
transcriptional regulation (see Chapter 21). Fluorescent proteins
produced in this way are said to be genetically encoded, which
simply means that cells can be induced to make an engineered
protein by adding in the appropriate DNA encoding it.
A common approach to making fluorescent fusion proteins
involves the green fluorescent protein (GFP). GFP is a fluores-
cent protein produced by the jellyfish Aequorea victoria, origi-
nally characterized by Osamu Shimomura and colleagues. In
1992, the gene for GFP was cloned. Shortly thereafter, GFP was
expressed in living organisms by Martin Chalfie and colleagues.
GFP fusions have since become a powerful tool in the arsenal of
cell biologists.
The basic approach to making a GFP fusion protein is shown
in Figure 19A-1a. DNA encoding GFP is fused to DNA cor-
responding to the exons of a gene of interest. The DNA is then
introduced into the cell or organism (methods for doing so in
specific cases are discussed in Chapter 21), which transcribes
the DNA into mRNA; the mRNA in turn is translated into a fluo-
rescent protein.
Depending on the protein of interest and its particular struc-
ture, the GFP tag can be added to either the N-terminus or the
C-terminus of the protein. If a functional assay is available, the
fusion protein is typically tested to ensure that the GFP tag
doesn’t adversely affect the protein’s function. The best test of
a fusion protein’s function is to see if it can replace the normal,
nonfluorescent protein in living cells.
If the GFP fusion protein can be expressed in the desired
cells, the location of the fluorescence will reveal the location
of the protein. Figure 19A-1b shows a C. elegans embryo that
is expressing a GFP-tagged protein that localizes to its cell-cell
junctions. The GFP fusion reveals the detailed location of the
tagged protein in a living embryo.
By making minor changes in the DNA of the GFP coding
region, and thus the resulting protein’s sequence, a number
of different color variants of GFP have been engineered—an
approach pioneered by Roger Y. Tsien and colleagues. These
different proteins absorb and emit light at a variety of
to one another to form the appropriate multisubunit proteins
or multiprotein complexes.
In addition to the preceding posttranslational events, some
proteins undergo a relatively unusual type of processing called
protein splicing, which is analogous to the phenomenon of RNA
splicing (discussed in Chapter 18). As you learned there, intron
sequences are removed from RNA molecules during RNA splic-
ing, and the remaining exon sequences are simultaneously
spliced together. Likewise, during protein splicing, specific ami-
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 Exon 1 Exon 2 Exon 3

5r Promoter 5r UTR Intron Intron GFP 3r UTR 3r

DNA
coding Start Stop
strand codon Transcription codon
Translation

Fusion N GFP C
protein
OR

Exon 1 Exon 2 Exon 3

5r Promoter 5r UTR GFP Intron Intron 3r UTR 3r

DNA
coding Start Stop
strand codon Transcription codon

Chapter 19  | Gene Expression: II. Protein Synthesis and Sorting

Translation

Fusion
N GFP C
protein

(a) Engineering a protein fused to GFP (b) C. elegans embryo expressing a 10 mm

GFP-tagged protein

Figure 19A-1  Creating Fusion Proteins. (a) Fusion proteins are created by combining DNA encod-
ing a protein of interest with DNA encoding another protein that can be visualized, such as GFP. The
GFP-encoding DNA is often placed in a position so that the resulting fusion protein contains GFP after
(that is, C-terminal to) or in front of (that is, N-terminal to) the rest of the protein. (b) A C. elegans em-
bryo expressing a GFP-tagged protein that localizes to cell-cell junctions (confocal microscopy).

wavelengths, thus producing a wide range of colors
(Figure 19A-2). Shimomura, Chalfie, and Tsien shared a
Nobel Prize for their work on GFP in 2008. In addition to GFP,
other fluorescent fusion proteins have been produced from
other organisms (such as coral), allowing cells to be engineered
to express a wide variety of fusion proteins simultaneously. By
using different wavelengths of fluorescent light, the locations
of multiple fluorescent proteins can be determined at the same
time in living cells.

QUESTION: Suppose you are using GFP fusion proteins

to study a cell-cell junction protein. How could you show that
a particular amino acid sequence in the protein is needed for
Figure 19A-2  Color Variants of GFP. targeting it to cell-cell junctions in cultured cells?

no acid sequences called inteins are removed from a polypeptide
chain, and the remaining segments, called exteins, are spliced
together to form the mature protein. Protein splicing is usually
intramolecular, involving the excision of an intein from a sin-
gle polypeptide chain by a self-catalytic mechanism. However,
splicing can also take place between two polypeptide chains
arising from two different mRNAs. For example, in some photo-
synthetic bacteria a subunit of DNA polymerase III is produced
from two separate genes, each encoding an intein-containing
polypeptide that includes part of the DNA polymerase subunit.
Once considered to be an oddity of nature, protein splicing has
now been detected in dozens of different organisms, prokary-
otes as well as eukaryotes.
CONCEPT CHECK  19-4
Enzymes can undergo allosteric regulation or regulation by
covalent modification (see Chapter 6). Which category do
posttranslational modifications fall into?
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