EXPRESSION OF THE FLUORESCENT PROTEIN mTurquoise2 IN THE PERIPLASM
OF Escherichia coli
Ira Handayani and Wien Kusharyoto*
Laboratory for Applied Genetic Engineering and Protein Design
Research Center for Biotechnology – Indonesian Institute of Sciences (LIPI)
Cibinong Science Center, Jl. Raya Bogor Km. 46, Cibinong-Bogor 16911
ABSTRACT
Many variants of cyan fluorescent protein (CFP) have been developed as fluorescent tags which are
widely used as donors in Förster resonance energy transfer (FRET) experiments. Recent
improvement of CFP variants resulted in mTurquoise2, a brighter variant with faster maturation,
high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a
monomeric fluorescent protein. Here, we describe the expression of mTurquoise2 targeted for
secretion via the general secretory (Sec) translocation pathway into the highly oxidizing periplasm
of Escherichia coli. The use of signal peptide MPB*1, a modified signal sequence of maltose
binding proteinwas investigated. The His6-tagged fluorescent protein was expressed in E. coli
NiCo21(DE3) and purified by means of immobilized metal ion affinity chromatography (IMAC) on
TALON™ matrix. In SDS-PAGE and Western blot analysis, a single band corresponding to a
molecular mass of approximately 28 kDa was observed, which correlated with the predicted
molecular mass based on the amino acid sequence of mTurquoise2.
Keywords:mTurquoise2, fluorescent protein, periplasm, Sec-pathway, translocon
*Corresponding author:
Tel. 021-8754587
Fax. 021-8754588
E-mail: [email protected]
1
INTRODUCTION
Since its discovery in 1962 fluorescent proteins (FPs) have become important tools to study
proteins and signaling events in living cells.[1,2] Due to their unique characteristic and properties,
they become a powerful toolkit for visualization of structural organization and dynamic processes in
living cells and organisms.[3,4,5]Many variants of fluorescent protein have been developed for
fluorescence- or foster-resonance energy transfer (FRET) applications to study protein-protein
interaction.[6,7]The most widely used for FRET experiment are Enhanced Cyan Fluorescent Protein
(ECFP).[8,9]Recent improvement of ECFP variants resulted in mTurquoise2, a brighter variant with
faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum
yield measured for a monomeric fluorescent protein.[10]
In the oxidizing environments such as the eukaryotic endoplasmic reticulum (ER) or bacterial
periplasm,
the
expression
of
FPs
can
impair
folding,
therefore
preventing
fluorescence.[11,12]Nevertheless, a study reported a successful expression of superfolder Green
Fluorescence Protein (sfGFP) in the periplasm of Escherichia coli (E. coli) via the Sectranslocon.[13]The Sec- translocase or translocon is the essential and ubiquitous system for protein
translocation across or into the membrane. The core channel, the SecYE complex, is conserved
across biological kingdoms and most of the polypeptide chains which are routed to extracellular or
membrane locations in bacteria use this pathway.[14]For the expression in E. coli periplasm maltose
binding protein (MBP) signal sequence (MBP*1) was successfully employed for efficient cotranslational translocation of sfGFP across the E. coli inner membrane.[13]
In many expression studies, proteins are designed to be expressed in the periplasm. This might be
due to the easiness of isolating proteins from this compartment than from whole cell lysates. In the
oxidizing environment of the periplasm the disulfide bond formation (Dsb)-system catalyzes the
formation of disulfide bonds, therefore, disulfide bond containing proteins, like antibody fragments
and many peptide hormones, are produced in the periplasm to enable folding into their native
conformation.[15,16]In order to reach the periplasm, the heterologous proteins are equipped with an
N-terminal signal sequence that guides them to the Sec-translocon, which is a protein-conducting
channel in the cytoplasmic membrane.[15,17]
1
Figure 1: Ribbon representation of a 3D model of the fluorescent protein mTurquoise2 with His6-tag at
the C-terminus. The chromophore is shown in balls-and-sticks representation.
Since the previous works had expressed the mTurquoise2 using a mammalian cell, here we
describe the first attempt to express mTurquoise2 (Fig. 1) into the highly oxidizing periplasm of E.
coli by targeting the secretion via the general secretory (Sec) translocation pathway using MBP*1
signal peptide.
MATERIAL AND METHODS
Design of the synthetic gene
The DNA sequence of the mTurquoise2 gene was codon optimized for expression in E. coli
using the Gene Designer software and DNA 2.0 algorithm (http://www.dna20.com). The gene
encoding mTurquoise2 was then synthesized by DNA2.0 (Menlo Park, CA, USA). The synthetic
gene was subsequently cloned into the expression vector pJ434 (T7-promoter, AmpR, pUC origin)
using appropriate restriction enzymes. The sequence of mTurquoise2 was equiped with MBP*1
signal peptide at N-terminus for translocation of mTurquoise2 into the periplasm of E. coli. At the
C-terminus of mTurquoise2, the sequence for six histidin (His6)-tag was included to facilitate
purification. The resulting vector is being referred to as pJE-mTurq2 (Fig. 2).
3
Protein Expression and Purification
The expression vector was transformed into host cells E. coli NiCo21(DE3) by heat-shock at
42C for 45 second. E. coli NiCo21(DE3) cells cointaining pJE-mTurq2 were grown on Luria
Bertani (LB) plates containing 100 µg/ml ampicillin. Single colonies were picked and grown
overnight in 10 ml LB medium containing 100 µg/ml ampicillin at 25ºC and 200 rpm in 500 ml
shaking flask to an OD600 of 0.8 - 1.0. IPTG (Sigma) was added after 6 h incubation, and growth
was continued for overnight. The cells were harvested by centrifugation (5000×g, 15 min, 4C) and
then resuspended in 50 mM phosphate-buffer (PBS) pH 7.2 containing 300 mM NaCl. Isolation of
mTurquoise2 extract from the periplasmic space was conducted by adding 20 mg/L lysozyme to the
protein extract and incubation on ice for 1 h. After centrifugation to remove cell debris, the
mTurquoise2 extract was subjected to protein purification. Purification of mTurquoise2 was
performed by immobilized metal-ion affinity chromatography (IMAC) on TALON™
chromatography matrix (Clontech).[18]One ml of the matrix were loaded into a PD-10 column (GE
Bioscience) and equilibrated with 50 mM PBS pH 7.2 containing 300 mM NaCl. Ten (10) mililiters
of periplasmic extract from 100 ml culture were applied to the column. After washing with 20 ml of
20 mM imidazole in 50mM PBS pH 7.2 containing 300 mM NaCl, fractions containing
mTurquoise2 were eluted from the column with 200 mM imidazole in 50 mM PBS pH 7.2
containing 300 mM NaCl. The protein expression and purification were verified by SDS-PAGE and
Western blot.
SDS-PAGE and Western Blot Analysis
The purified protein fractions were prepared for SDS-PAGE analysis by mixing one part of
sample with one part of Laemmli sample buffer (Biorad). Samples were analyzed by 15% SDSPAGE. Proteins were visualized by Coomasie blue R-250 staining or proteins were transferred to
nitrocellulose membrane (Biorad) for immunoblotting. The mTurquoise2 fragment was detected by
His Detector™ Nickel-HRP (KPL) and colometric detection with tetramethylbenzidine as substrate
according to manufaturer’s protocol
4
Results and Discussion
In studying the signaling events, fluorescent biosensors have become important tools with high
spatial and temporal resolution in living cells. Biosensors are genetically engineered constructs that
report on protein conformation or activation status by alterations in spectral properties. FRET
(Fluorescence- or Förster- Resonance Energy Transfer) is particularly popular in such
sensors.[5]Cyan variants of green fluorescent protein are widely used as donors in Förster resonance
energy transfer experiments. Recently, the Cyan Fluorescent Protein (CFP) has been improved to
produce brighter variant such as mTurquoise. The use of mTurquoise offers some benefits such as
improved brightness, optimized folding, mono-exponential life time, excellent photostability, and
high-fluorescence quantum yield.[10]
A significant subset of proteins in eukaryotes needs to be exported to their site of action from
the cytosol, the usual site of protein synthesis. Many of these exported proteins are synthesized in a
precursor form with an additional N-terminal amino acid extension called a signal peptide.[19]In
order to enable the expression of the mTurquoise2 fragment in the periplasm and to translocate
mTurquoise2 via the SecYEG translocon into the the highly oxidizing environment of E. coli
periplasm, a single sequence MBP*1, which has been used for effective translocation of other
protein fragments in the periplasm of E. coli[14],was added at the N-terminus of mTurquoise2
construct. At the C-terminus of the mTurquoise2 construct, a sequence for six histidin (His6)-tag
was included to facilitate purification (Fig. 2). Secretory production of recombinant proteins
provides several advantages, such as the N-terminal amino acid residue of the secreted product can
be identical to the nature gene product after cleavage of the signal sequence by a specific signal
peptidase.[20]In addition, recombinant protein purification is simpler due to fewer contaminating
proteins in the periplasm.
Figure 2:
Schematic presentation of the expression vector pJE-mTurq2 harbouring the syn-mTurquoise2
gene for the expression in E. coli. The signal sequence MBP*1 was used to direct the protein
translocation into the bacterial periplasm. His6 was used to facilitate protein purification.
5
In order to develop robust expression of a recombinant mTurquoise2 in the E. coli periplasm, a
synthetic gene encoding mTurquoise2 was designed by using codons optimized to reflect
abundantly translated E. coli mRNAs. The codon usage of the gene encoding mTurquoise2
exhibited almost 11% of codons showed rare occurrence in E. coli genes. Rare codons and poor
codon bias may be a possible cause of an inefficient translation and protein production in E. coli. To
overcome this limitation, the gene encoding mTurquoise2 was adapted to the codon bias of E. coli
genes with the help of Gene Designer software[21]for increased soluble expression in E. coli.
Figure 3: Codon optimization of the gene encoding the fluorescent protein mTurquoise2 for expression in
E. coli. Sequence alignment of native or wild-type (WT) and codon-optimized gene (Opt) is
shown. A coding sequence for the signal peptide MBP*1 at 5’-end is underlined and a coding
sequence for His6-tag is shown at 3’-end. The rare codons are marked in red boxes. AA: the
amino acid sequence
6
Codon usage preference in a gene is often indicated by a higher value of codon adaptation index
(CAI). The genes designed to match host bias or obtained by maximizing CAIvalue have been
expressed successfully in different studies.[22]The CAI was calculated by using online tool available
in the web.[23]The native gene encoding mTurquoise2 exhibited a CAI-value of 0.67, whereas the
designed gene resulted in an increase of CAI to 0.83. The designed gene encoding His6-tagged
mTurquoise2 was subsequently synthesized and cloned into the expression vector pJExpress 434,
resulted in an expression casette pJE-mTurq2.
The resulted expression vector pJE-mTurq2 harboring the synthetic gene was subsequently
verified by enzyme digestions using the restriction enzymes NdeI and NdeI/XhoI. The results
indicated that the expression vector pJE-mTurq2 exhibited the correct size of approximately 4900
bps after digestion with NdeI. Double digestion with NdeI and XhoI yielded a fragment with the size
of approximately 4150 bps and the fragment of the synthetic gene encoding His6-tagged
mTurquoise2 with the size of 750 bps as previously designed (Fig. 4).
Figure4:
Verification for the presence of syn-mTurquoise2 gene in the expression vector pJE-mTurq2 by
enzyme digestion. Digestion with NdeI resulted in a linearized vector with the size of
approximately 4900 bps. Double digestion with both NdeI and XhoI yielded a DNA band with
the size of around 750 bps for the syn-mTurquoise2 gene.
7
For the production of mTurquoise2, the expression vector pJE-mTurq2 was transformed into E.
coli NiCo21(DE3) by heat-shock. The recombinant protein was expressed in E. coli cells by
induction with IPTG at 25ºC three hours after inoculation into the culture medium. Upon protein
translation in the cytosol of E. coli the polypeptide of the His6-tagged mTurquoise2 with the signal
peptide MBP*1 was transported across the inner membrane, where the signal peptide was cleaved
by the signal peptidase, and the His6-tagged mTurquoise2 was translocated into the E. coli
periplasma, where it underwent further protein folding.
After centrifugation, the cell pellet was resuspended and subsequently treated with lysozyme to
facilitate the release of only proteins in the periplasm of E. coli. Periplasmic protein extracts having
His6-tag were purified by IMAC on TALON™ resin. Upon verification by SDS-PAGE (Fig. 5 A)
and Western blot (Fig.5 B), the recombinant mTurquoise2 appeared as a single band at
approximately 28 kDa with relatively high purity, which corresponded to the calculated molecular
weight of the protein based on the amino acid sequence of mTurquoise2 with its C-terminal His-tag.
As depicted by the SDS-PAGE and Western blot results, the expression level of the recombinant
mTurquoise2 was rather low compared to the expression level of other proteins in the periplasm.
Comparion with a serie of bovine serum albumin (BSA) concentrations on SDS-PAGE gel (data not
shown) revealed that the amount of the expressed protein was approximately 1 mg protein per liter
E. coli culture.
Figure 5: Verification of expression and purification of mTurquoise2. (A) SDS-PAGE analysis of
mTurquoise2 isolated from the periplasmic space of E. coliNiCo21(DE3). Lane M: protein
marker; lane CE: periplasmic extract of E. coliNiCo21(DE3); lane FT: flow through of column
containing Talon™ resin; lane W1 and W2: fractions from washing steps; lane E1–E3: fractions
from elution steps of purified mTurquoise2. (B) Western blot analysis of the purified
mTurquoise2 from elution fraction E3.
8
In previous attempt to form a green fluorescent fluorophore in the periplasm via the SecYEG
translocon, a bacterial expression construct containing ssMBP-sfGFP with a native signal sequence
of MBP was created and the expressed recombinant protein was accumulated primarily in the
cytoplasm of E. coli.[24]On the other hand, Lee and Bernstein described an MBP signal sequence
(MBP*1) that contains three amino acid mutations, optimized for efficient co-translational
translocation across the E. coliinner membrane.[25]Here, we exploited the signal peptide MBP*1 for
the translocation of mTurquoise2 into the bacterial periplasm, and showed that mTurquoise2 could
be expressed in the periplasm of E. coli. However, it remains unclear whether mTurquoise2 could
correctly fold and fluoresce in such an oxidizing environment in E. coliperiplasm.
Further works are still necessary in order to increase the expression level of the recombinant
mTurquoise2, for instance by changing the expression vector and/or the signal sequence, and to
enable expression of actively fluorescent, SecYEG translocated mTurquoise2 for the bacterial
periplasm.
CONCLUSION
A strategy for the preparation of a monomeric enhanced cyan fluorescent protein mTurquoise2 in
the periplasm of E.coli has been established. The signal peptide MBP*1 has been successfully
employed to correctly localize the protein into the E. coli periplasm. The protein mTurquoise2 can
be further modifiedby mutational experiments in the generation of a rapidly folding and robust
stable mutants of fluorescent proteins like those previously described for EGFP.
ACKNOWLEDGEMENTS
This work was supported by a grant from Competitive Research Program of the Indonesian Institute
of Sciences (LIPI).
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