Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro

Article (PDF Available)inNature Protocol 1(2):714-9 · February 2006with25 Reads
DOI: 10.1038/nprot.2006.114 · Source: PubMed
Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires approximately 3 d, and their conjugation to oligonucleotides requires 1-4 h.
Profluorescent protein fragments for fast bimolecular
fluorescence complementation in vitro
Vadim V Demidov & Natalia E Broude
Center for Advanced Biotechnology, Boston University, 36 Cummington Street, Boston, Massachusetts 02215, USA. Correspondence should be addressedto
V.V.D. ( or N.E.B. (
Published online 6 July 2006; doi:10.1038/nprot.2006.114
Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a
preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly
becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment
is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from
inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are
linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few
minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires B3d,andtheirconjugation
to oligonucleotides requires 1–4 h.
Bimolecular complementation of fragments of fluorescent proteins
is a robust assay for directly monitoring conformational changes
or interactions of proteins, and for detecting specific nucleic
acid sequences
. The approach is based on the reassembly of a
fluorescent protein from two nonfluorescent fragments driven by
additional biomolecular interactions and resulting in restoration of
fluorescence. In all prior studies, fluorescent chromophore formed
de novo within the reconstituted full-size protein (Fig. 1a). There-
fore, it took hours to restore the fluorescence of a split fluorescent
protein, as formation of a protein chromophore is generally known
to be a slow process
In the course of our recent study on protein fluorescence
complementation triggered by nucleic acid complementary inter-
actions, we unexpectedly observed rapid kinetics of fluorescence
. These results suggested that the large N-terminal
fragment of split EGFP used in this study contained a preformed
chromophore. Computer simulations verified that the large EGFP
fragment may develop a compact structure in which arrangement
of the chromophore-forming amino acids is essentially the same as
in the full-size EGFP
. These data indicated that, under certain
conditions, the protein chromophore may spontaneously form
within the large N-terminal EGFP fragment, which will instantly
become brightly fluorescent upon complementation with the
C-terminal EGFP fragment (Fig. 1b).
We describe here the protocol that enabled us to isolate the large
EGFP fragment with a preformed chromophore. Using the chro-
mophore-containing large EGFP fragment yields a fluorescent
response within 1 min, when reassembly of a split protein is
supported by DNA hybridization
. The proposed methodology
therefore represents a promising alternative for the in vitro mon-
itoring of biomolecular interactions in real time, when compared to
competing fast-responding techniques: protein-based fluorescent
resonance energy transfer (FRET)
and nucleic acid–based mole-
cular beacons
. Indeed, the reassembled split EGFP generates a
much stronger signal over background than the protein-based
FRET. Another potential advantage is the expected reduced sensi-
tivity of a protein-shielded chromophore inside the reassembled
split EGFP to environmental changes, such as pH, solvent and salt
concentration, as compared to the solvent-exposed molecular
protein fragments
De novo formed
Preformed 'dark'
Figure 1
The principle of protein fluorescence complementation. Fluorescent
protein is split into two nonfluorescent protein fragments, which are tagged
by interacting molecules A and B (A and B could be proteins or, as in
experiments shown in Figure 4, oligonucleotides). Binding of A to B brings
the nonfluorescent protein fragments in close proximity that reconstitutes
split protein and restores fluorescence. (a) Traditional approach with slow
development of fluorescence. Protein fragments do not have a fluorogenic
chromophore prior their complementation; it slowly (up to 100 min) forms
afterwards. (b) An unconventional approach presented here and yielding a
rapid signal response. One of the protein fragments forms a fluorogenic
chromophore before reassembly. This profluorescent polypeptide should rapidly
develop fluorescence upon complementation with another protein fragment.
VOL.1 NO.2
© 2006 Nature Publishing Group
Experimental design
Our experimental design is shown schematically in Figure 2.
Specifically, the DNA sequences encoding the large (N-terminal)
and small (C-terminal) EGFP fragments were cloned in the
pTWIN-1 vector to yield the C-terminal fusions with the Ssp
DNAB intein. The N- and C-terminal EGFP-coding sequences
were obtained by PCR amplification of the corresponding frag-
ments of the pEGFP-1 vector containing the gene encoding EGFP.
Each PCR product was ligated into the SapI/PstI-digested pTWIN-
1 vector using adaptors; this cloning eliminated a second intein.
With the chosen PCR primers (see MATERIALS), the large EGFP
fragment contained the 158 N-terminal amino acids of EGFP plus a
C-terminal cysteine, whereas the smaller fragment contained the
remaining 81, C-terminal amino acids of EGFP plus an N-terminal
cysteine. Terminal cysteines were added to the protein fragments to
facilitate their conjugation to oligonucleotides (see ANTICIPATED
RESULTS). The N- and C-terminal locations of added cysteines in
the small and large EGFP fragments, respectively, warrant their
reassembly similar to native conformation.
Protein fusions were expressed in E. coli,followedbytheir
isolation from bacterial cells as inclusion bodies. After solubilizing
and refolding these fusions from inclusion bodies, both EGFP
fragments were cleaved from intein and purified using chitin
columns. Intein strongly binds to chitin beads to anchor the protein
fusion to a column, and it remains on the column after slow self-
splitting from the fused protein triggered by a change of the buffer
pH from 8.5 to 7.0. The flowchart of the entire procedure is shown
in Figure 3. It should be noted that expression of the N-terminal,
large EGFP fragment fused to intein is usually less efficient than
expression of the C-terminal, small EGFP fragment-intein fusion.
Therefore, larger cell-culture volumes are recommended for grow-
ing cells expressing the N-terminal EGFP fragment for obtaining an
amount comparable with the amount of C-terminal fragment.
This procedure yields the large EGFP fragment with a preformed
chromophore, which is revealed in: (i) a weak but characteristic
fluorescence of the isolated N-terminal protein fragment, and (ii)
the fast development of strong fluorescence upon addition of the
C-terminal protein fragment, provided that both EGFP fragments
are equipped with additional interacting molecules A and B
(Fig. 1). Alternatively, A and B can interact with a third molecule,
C, which will juxtapose profluorescent protein fragments (not
shown). Here, we used a pair of complementary oligonucleotides
for EGFP complementation. EGFP fragments can also be fused to
the protein partners to detect their cross-interaction or conforma-
tional change induced by the binding of a small ligand.
Primers used to PCR-amplify coding sequences of large and small EGFP
fragments: Large EGFP fragment with C-terminal cysteine; Primer
Small EGFP fragment with N-terminal cysteine; Primer BETA-CYS_dir:
pEGFP-1 or other plasmid containing the EGFP coding sequence
pTWIN-1 vector (New England Biolabs)
BL21(DE3) pLys competent E. coli cells (Stratagene)
Luria-Bertani (LB) cell-culture media
Isopropyl thiogalactoside (IPTG, 1 M stock)
Cell resuspension buffer: 50 mM Tris-HCl (pH 8.5), 25% sucrose, 1 mM
EDTA, 10 mM 1,4-dithiothreitol (DTT)
Inclusion bodies washing buffer: 50 mM Tris-HCl (pH 8.5), 0.5% Triton
X-100, 100 mM NaCl, 1 mM DTT, 1 mM NaEDTA
N- or C-terminal
split EGFP gene
CDB Intein 1
CDB Intein 1
Cloning & expression
Intein 1
N-EGFP fragment
C-EGFP fragment
Intein 2pTWIN1
T7 promoter
C-EGFP fragment
N-EGFP fragment
Chitin binding and pH-
induced cleavage of intein 1
Figure 2
Experimental design used here for cloning and isolating the two
fragments of split EGFP. CBD, chitin-binding domain; MCS, multiple cloning site.
Cloning of EGFP fragment-intein fusions
Overexpression of proteins
in inclusion bodies
Solubilization and refolding
of protein fusions
Binding of protein fusions
to chitin columns
Fusions self-splitting with
recovery of EGFP fragments
SDS-PAGE protein analysis
Spectrophotometric analysis
of the large EGFP fragment
th, nm
400 500
EGFP fragments
Small M
Figure 3
Flowchart of the major procedure described in this protocol.
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© 2006 Nature Publishing Group
Protein solubilization buffer: 25 mM MES (pH 8.5), 8 M urea, 10 mM
NaEDTA, 0.1 mM DTT
Protein refolding buffer: 50 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM DTT
Protein refolding buffer (neutral version): same composition as the previous
buffer but with pH 7.0
Column equilibration-loading buffer: 50 mM Tris-HCl (pH 8.5), 500 mM
NaCl, 1 mM EDTA, 1 mM PMSF and 0.1% Triton X100
Column cleavage buffer: same composition as the previous buffer but with
pH 7.0
Precast 15% polyacrylamide gels for protein electrophoresis (PAGE,
Tris-glycine type, Cambrex)
Protein electrophoresis buffer: 25 mM Tris, 0.2 M glycine, 0.1% SDS (pH 8.3)
Chitin beads (New England Biolabs, cat. no. S6651)
Thermal cycler (PTC-100 or PTC-200, MJ Research)
Controlled-environment incubator shaker (model G25, New Brunswick
Superspeed centrifuge (SORVALL RC 5B, Thermo Electron Corporation)
Sonifier cell disrupter (model W185c, Branson Sonic Power)
Poly-prep chromatography columns (Bio-Rad, cat. no. 731-1550)
Set of Pipetmans (P2 to P1000, Gilson)
Vertical electrophoresis system (10 10 cm, Fisher Biotech)
Spectrofluorometer (Hitachi F-2500) and, optionally, spectrophotometer
(Hitachi U-3010) with quartz cuvette(s)
1| PCR-amplify DNA sequences coding for the large and small EGFP fragments using a common PCR practice
(, and using a plasmid with the EGFP gene (Clontech) as a template and the
corresponding primers (see MATERIALS and Experimental design).
2| Separately clone each of the corresponding DNA sequences into pTWIN-1 vector using traditional recombinant techniques
to yield the IPTG-inducible fusions of EGFP fragments to mini-intein.
m CRITICAL STEP Check the cloned plasmid sequences for correctness of all necessary protein-expression elements
(promoter, initiation and stop codons, and protein-coding genes).
3| With these plasmids, transform the protease-deficient BL21 (DE3) pLys competent E. coli cells.
4| Grow the transformed E. coli cells in a shaking incubator overnight at 37 1C in 2 ml LB medium.
5| By taking the corresponding aliquots, dilute the cell culture 500-fold in 200 ml to 1 liter LB (for submilligram to milligram
protein amounts), and allow cells to grow at 37 1C for about 2–3 h up to A
E 0.6.
6| Induce cells with 0.35 mM IPTG and grow them overnight at 25 1C or for 4 h at 30 1C (in the latter case, somewhat better
protein expression is normally observed).
m CRITICAL STEP Check the efficiency of IPTG induction by SDS-PAGE directly loading onto a 15% polyacrylamide gel a required
amount of SDS-denatured cell suspension before and after induction, and analyzing the intensity of corresponding protein bands
(B35 kDa and B40 kDa for the intein-fused small and large EGFP fragments, respectively).
7| Precipitate cells by centrifugation at 15,000 r.p.m. for 10 min at 4 1C. Resuspend cells in resuspension buffer (15 ml/1 l
cell culture).
8| Freeze 1-ml aliquots at –70 1C for 10 min, then thaw them at 37 1C for 5 min; repeat the freeze-thaw process three times.
9| Keeping cells on ice, and break them up by sonication with three 30-s bursts, each followed by a 30-s interval.
10| Combine the resulting samples and precipitate the inclusion bodies by centrifugation at 15,000 r.p.m. for 5 min at 4 1C.
11| Remove the supernatant, resuspend the precipitate in washing buffer (10 ml/1 l cell culture) by vortexing and sonicate
this suspension as before.
m CRITICAL STEP Keep this supernatant in a refrigerator until the end of the procedure to use it as an alternative source
for isolation of EGFP fragments from a soluble cellular fraction (see the TROUBLESHOOTING TABLE, Step 14).
12| Centrifuge for 5 min at 15,000 r.p.m. and repeat the washing step three times, each time discarding the supernatant.
13| Resuspend pellets in solubilization buffer (5 ml/1 l cell culture) and incubate at 25 1C for 1 h. Clear the solution by
centrifugation at 15,000 r.p.m. for 5 min.
14| Use the supernatant containing solubilized denatured protein to refold it by adding this solution drop by drop to an
excess of the refolding buffer with 1:100 dilution. For drop-by-drop dispensing (normally requires several minutes), use a 100-ml
Pipetman (P100) with B1-s intervals between drops.
PAUSE POINT Leave the refolding solution overnight at 4 1C.
m CRITICAL STEP We believe that intein facilitates the proper folding of EGFP fragments
. Still, the success rate of correct
refolding, and hence of chromophore formation, is B50%: we got two fluorescently active samples of the large EGFP fragment
out of four independent isolations from inclusion bodies (with a smaller yield, a fluorescently active preparation of split EGFP
VOL.1 NO.2
© 2006 Nature Publishing Group
was also obtained from a soluble cellular fraction by an alternative protocol without refolding). So, refolding of several samples
in parallel is recommended at this step of the protocol. It is also recommended to estimate the concentration of solubilized
proteins by SDS-PAGE using protein standards. The protein concentration 4100 mg/l would be optimal for subsequent protein
refolding and column purification.
15| Fill an empty 9-cm-high, conical 0.8 4 cm polypropylene chromatography column with 2 ml of the chitin bead
suspension (supplied as a slurry in 20% ethanol with a bead content of B50% (vol/vol)). Equilibrate the column with 10 ml
of the column equilibration-loading buffer.
m CRITICAL STEP For the intein-fused protein purification using the chitin column procedure, see http://t7l.bimcore.¼’chitin%20column%20new%20england%20biolabs’ and
16| Load a refolded protein solution onto the column and wash it with 20 ml of the column equilibration-loading buffer.
17| Wash the column with 10 ml of the cleavage buffer and close the bottom tip with the tip closure.
18| Load the column with 10 ml of the cleavage buffer, then close the top with the end cap.
PAUSE POINT Leave the column at room temperature overnight for intein self-cleavage.
19| Open the column’s top end and the bottom tip to elute EGFP fragment split from intein. Collect B300-ml fractions and
analyze them by SDS-PAGE for purity and amounts of the large and small EGFP fragments (B18 kDa and B10 kDa, respectively;
see Fig. 3). Close and save the column for possible additional elution. Store both protein solutions at 4 1C.
20| Verify the presence of the chromophore within the large EGFP fragment by spectrophotometry. The chromophore-containing
protein fragment should show significant absorbance in the range of 300–400 nm. The presence of a chromophore in the large
EGFP fragment should be more evident by recording its fluorescence with excitation maximum near 350 nm and emission
maximum near 450 nm (see Fig. 3). The fluorescence intensity of this protein fragment should be B100 times weaker than
the peak fluorescence of the intact EGFP
Steps 1–3: 2–3 d
Steps 4–6: 20–30 h
Steps 7 and 8: 1 h
Steps 9–13: 2 h
Step 14: 15 h
Steps 15–18: 16–17 h
Step 19: 3 h
Step 20: 30 min
The protein-oligonucleotide or protein-protein conjugation reactions normally require 1–4 h, depending on the particular
chemistry used.
Troubleshooting advice can be found in Table 1.
Troubleshooting table.
Step 6 Little or no protein expression was detected
after induction.
Vary the IPTG concentration and/or cell growth temperature to find the optimal
conditions for the particular cell clone. If this does not help, grow a single clone of
the transformed E. coli cells first on the agar plate, then repeat Steps 4–6.
Step 14 Concentration of the refolded large EGFP-intein
fusion is low (o1mg/l
In this case, subsequent column purification of the large EGFP fragment will be
inefficient. As an alternative to the column-splitting procedure, cleave the fusion
directly in the refolding buffer with pH 7.0 overnight at 4 1C. In some applications,
there is no need for separation of the large EGFP fragment from the intein, as it will
not interfere with the downstream procedures. If the intein removal is necessary,
the 25-kDa split intein can be separated from solution of the 18-kDa large EGFP
fragment by size- selective ultrafiltration using Centricon centrifugal filter
microdevices (YM-50, Millipore). Alternatively, fluorescently active EGFP fragments
VOL.1 NO.2
© 2006 Nature Publishing Group
Profluorescent protein fragments with mature chromophore should be useful for detecting many types of fast
pairwise interactions. To demonstrate this potential, we obtained the chromophore-containing large and the
complementary small EGFP fragments from the inclusion bodies (see Fig. 3), and used them in the protein fluorescence
complementation assay triggered by the DNA-DNA duplex formation
. The DNA duplexes normally form very quickly
Given that refolding and fluorescence restoration of the chromophore-containing full-size EGFP from totally unfolded
state requires just a few minutes
, we expected that reassembly of the split EGFP from fragments, one of which
already contains the chromophore, should result in the fast development of protein fluorescence, if supported by
DNA hybridization.
In our in vitro system
, large and small EGFP fragments iso-
lated using the above procedure were first biotinylated using
sulfhydryl-based chemistry, then coupled with complementary
biotinylated oligonucleotides using streptavidin as a linker.
The oligonucleotide-conjugated large EGFP fragment was
only weakly fluorescent, and the small EGFP fragment-
oligonucleotide conjugate did not show any detectable fluores-
cence. However, when the two EGFP fragments were brought
together by nucleic acid complementary interactions, a strong,
up to 100-fold increase in fluorescence was detected with exci-
tation-emission spectra resembling EGFP and with a t
r 1
min (Fig. 4)
. The fluorescence intensity of the reassembled
complexes varied from experiment to experiment, with
maximal response close to that of the intact EGFP. In contrast,
control experiments (i.e., mixing streptavidin-bound protein
fragments without complementary oligonucleotides) did not
show any appreciable increase in fluorescence. These proof-
of-principle data demonstrate that protein fluorescence
complementation with the preformed chromophore occurs
quickly. We believe that this approach could be extended
to other fluorescent proteins
, to other conjugation
chemistries and to detecting various complexes in vivo by
injecting the corresponding conjugates into different cellular
ACKNOWLEDGMENTS We acknowledge our graduate students P. Chalasani and
H.-W. Yiu, as well as C. Witte-Hoffmann, I. Smolina and Y. Yu, who participated
in the protocol development. We also thank C.R. Cantor for inspiration and
encouragement in this project.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at
Reprints and permissions information is available online at
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Split reassembled EGFP
Split EGFP
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500 550
Wavelength (nm)
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Figure 4
Fluorescent response kinetics of the split EGFP system upon DNA
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    • "Reassembly of the fluorescent protein takes place in a narrow set of conditions and with great dynamic range that results in high signal/background ratio [8]. These features of protein complementation make them perfect for both protein and nucleic acid studies1231011121314. Recently, we described a method for RNA visualization in live cells that uses a combination of fluorescent protein complementation and high affinity interaction of an RNA-binding protein with an aptamer123. "
    [Show abstract] [Hide abstract] ABSTRACT: Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio.
    Full-text · Article · Dec 2011
    • "An implicit condition of these systems is that the two halves do not reconstitute by themselves and that only the interaction of protein 'X' with protein 'Y' (where X and Y are fused to the split domains of an FP, respectively) triggers the reconstitution of an FP. It is known, however, that the large N-terminal fragment of split-GFP can pre-form a chromophore under certain conditions (Demidov and Broude, 2006). Moreover, certain fragment combinations can spontaneously reconstitute in the absence of attenuators (Cabantous et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597-609). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.
    Full-text · Article · Mar 2008
  • [Show abstract] [Hide abstract] ABSTRACT: This critical review describes our current knowledge on the folding, stability and conformational dynamics of fluorescent proteins (FPs). The biophysical studies that have led to the elucidation of many of the key features of the complex energy landscape for folding for GFP and its variants are discussed. These illustrate some important issues surrounding how the large beta-barrel structure forms, and will be of interest to the protein folding community. In addition, the review highlights the importance of some of these results for the use of FPs in in vivo applications. The results should facilitate and aid in experimental designs of in vivo applications, as well as the interpretation of in vivo experimental data. The review is therefore of interest to all those working with FPs in vivo (103 references).
    Full-text · Article · Oct 2009
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