Summary
Fluorescent proteins are crucial and widely used tools in many fields of the life sciences. Nowadays, there is a great range
of different fluorescing proteins with optimized properties for certain applications. In our project, we chose to use
mCherry as a marker protein, because of its fast maturation time, its bright fluorescence, and the possibility to detect
mCherry expression with the naked eye. Because mCherry is not very well characterized in the Parts Registry, we decided
to add some data to it. We expressed and purified mCherry comparing different purification protocols and measured
fluorescence- and absorbance-spectra, as well as the fluorescence intensity normalized to Texas Red. Thus, we introduce a
measurement standard to measure mCherry fluorescence in relative fluorescence units (RFU). Additionally, we examined the
pH-sensitivity and the tolerance to light-exposure of mCherry.
Since the first successful cloning of the green fluorescent protein encoding gene GFP of Aequorea victoria in 1992
(Prasher et al. 1992) fluorescent proteins became a widely used tool in many fields of research. In contrast
to antibodies labeled with fluorophores that must cross the cellular membrane disturbing the cellular integrity,
fluorescent proteins enable live cell imaging and the investigation of native states of the cell.
Because of the wide range of applications for fluorescing proteins there was a great interest in discovering and
engineering improved variants and a wider color spectrum. In the last few years, red fluorescent proteins became
more and more important. Common native red fluorescing proteins are often dimeric or tetrameric, hinder their usage
in experimental setups . Directed mutation of dsRFP from the corallimorpharia Discosoma sp. led to the
first monomeric red fluorescing protein mRFP1. Unfortunately, the mutations decrease the quantum
yield and photostability (Shaner et al. 2004). During further protein engineering attempts, scientists were able to
create the red fluorescent protein mCherry. mCherry is a 26.7 kDa protein that shows a short maturation
time of about 15 minutes and a low acid sensitivity. Its excitation maximum is at 587 nm and it
has an emission maximum at 610 nm (www.fpbase.org). In 2006, the crystal structure of mCherry was published (Shu and Remington 2006).
Crystal structure of mCherry.
mCherry consists of 13 beta-sheets which form a beta-barrel and three alpha helices. The chromophore is made of methionine,
tyrosine and glycine which posttranslationally form an imidazolinone (Shu et al. 2006).
Expression
To further characterize mCherry in vitro, we purified mCherry comparing two different purification
protocols.
For the IMPACTTM kit we cloned mCherry (BBa_J06504) into the purification and expression vector pTXB1 from NEB and, for the his-tag purification, in pSB1C3 adding six histidines to the C-terminus of mCherry ( BBa_K2926048 ). Both expression vectors were transformed
in Escherichia coli ER2566. After induction with IPTG both cultures showed the characteristic red color of mCherry
expressing bacteria (Fig. 2 and Fig. 3).
Expression cultures of mCherry encoded by pTXB1 (left) and mCherryHis encoded by pSB1C3 (right).
Expression cultures of mCherry in pTXB1 and mCherryHis in pSB1C3 in E. coli ER2566 were cultivated to an OD of
around 0.6 at 37 °C in LB with 100 mg Ampicillin per L. Protein expression was induced by addition of IPTG to a final
concentration of 0.4 mM. After additional 30 min at 37 °C cultures were transferred to 17 °C and protein expression
was performed over night.
The expression culture of mCherry in pTXB1 showed a brighter red color which indicates a higher expression level.
Harvested expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).
Expression cultures of mCherry and mCherryHis in E. coli ER2566 were harvested by centrifugation at
4 °C for 20 min and 4 000 rpm.
Purification columns for IMPACTTM- (left) and Ni-TED-purification (right).
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 000 rpm for 15 s. The lysate
was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm. Cleared lysate was loaded onto a chitin column
(IMPACTTM-purification) or a Ni-TED column (purification via his-tag) and washed with wash buffer. Finally, the protein
was eluted, washed with PBS, and concentrated.
A Bradford assay revealed that expression and purification using the IMPACTTM-kit resulted in a higher yield since
we were able to purify 985 µg mCherry from a cell mass of 2.13 g compared to 39.4 µg mCherryHis from a cell mass of 1.92 g.
Both purification methods were analyzed on a SDS-PAGE (Fig. 5).
SDS-PAGE of the protein purification. E. coli lysate of the expression culture,
flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98 °C
for 10 min in SDS-PAGE loading buffer containing DTT, and loaded on an polyacrylamide-gel (12 %). The proteins
were separated through electrophoresis (25 mA). Suggested mCherry bands in the lane with purified proteins were
marked in red.
The SDS-PAGE shows an intense band at the estimated height of around 27 kDa in every lane. This indicates that mCherry
as well as mCherryHis have successfully been expressed and purified. The bands in the wash- and flow-through-fraction of the column-purification show, that not
all the proteins efficiently bind to the purification columns.
In the last lane it is visible that we were able to purify mCherry as well as mCherryHis. While the IMPACTTM
purification
resulted in a higher yield, the purity of mCherryHis was higher as the purified protein lane in Fig. 5 indicated.
Following the SDS-PAGE we analyzed the purified
protein via MALDI-ToF. For this purpose, we excised the marked bands (Fig. 5)
from the SDS-PAGE and started a tryptic digestion of the washed gel fragment. Analysis via MALDI-ToF confirmed that we were
able to purify mCherry (Fig. 6).
Mass spectrum of mCherry (1) and mCherryHis (2) after tryptic digestion.
Excised bands from the SDS-PAGE of mCherry and mCherryHis were washed, digested over night with trypsine and
co-cristallyzed with a α-Cyano-4-hydroxycinnamic acid-matrix on a MALDI target. The mass spectrum was
recorded in a MALDI-ToF MS from Bruker
Daltronics and data was evaluated using the software BioTools (Bruker).
The generated mass spectra and mass lists were evaluated using the software BioTools. To compare the experimentally
determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected
protein
sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match both proteins
to the theoretical spectrum. Additionally, we were able to detect the his-tag from mCherryHis in the mass list.
Characterization
To gain some more knowledge about mCherry we analyzed different properties of the protein. First, we measured its
emission- and absorbance spectra (Fig. 7).
Emission- and absorbance spectra of mCherry. Emission- (dashed lines) and absorbance-spectra
(solid lines) of mCherry purified via IMPACTTM-kit (dark purple) and his-tag (pink) were measured (λEx=570 nm,
λEm=600 nm to 850 nm) using the TECAN infinite M200 and normalized to their maximum.
The resulting spectra show, that adding a his-tag to mCherry does not alter the emission- or excitation spectrum of mCherry.
The excitation maximum of mCherry is at 587 nm, the emission maximum at 608 nm.
Next, we compared the fluorescence intensity of the two different mCherry-variants normalized to Texas Red (Fig. 8).
Fluorescence intensity of the dilution series of the two mCherry variants. Fluorescence intensity of
a dilution series of mCherry purified via IMPACTTM-Kit (dark purple) and mCherryHis (pink) was measured
(λEx=570 nm, λEm=610 nm) using the TECAN infinite M200. Furthermore, we normalized it to the fluorescence intensity of 0.5 µM Texas Red
at the same wavelength.
The fluorescence intensity of mCherryHis seemed to be higher than the intensity of mCherry purified via IMPACTTM kit.
This might be due to the different purification protocols. Cleavage of mCherry from the chitin column during the
IMPACTTM-purification is mediated through incubation of the column for 20-24 h in DTT at room temperature. Those
purification conditions might have a negative impact on the protein. Compared to Texas Red, the fluorescence intensity of
1 µmol mCherryHis equals the fluorescence intensity of 1.92 µmol of the fluorescent dye. In contrast, the fluorescence
intensity of 1 µmol mCherry purified via IMPACTTM protocol equals the fluorescence intensity of 565 nmol Texas Red.
Additionally we wanted to characterize the light tolerance and the pH-range of mCherry to get insight into the
optimal handling procedures. To determine the stability of mCherry it was exposed to normal daylight in the lab at
room temperature for three hours (Fig. 9).
Fluorescence intensity of mCherry in dependence on light exposure. mCherry was exposed to normal
daylight at room temperature. The remaining fluorescence intensity was measured at determinated time points (λEx=570 nm,
λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200. Furthermore, we normalized it to the intensity at t=0.
The results show that there is no significant decrease in the fluorescence intensity within the first 30 minutes.
After 2 h the fluorescence intensity already decreases more than 50 %. Due to the determined photostability
we suggest storing mCherry in the dark as long as possible to avoid photobleaching.
An often stated advantage of mCherry is its low acid sensitivity. To analyze the pH-range of mCherry, we
measured the remaining fluorescence intensity after incubation in buffers with different pH-values (Fig. 10).
Effect on the pH on the fluorescence intensity of mCherry. mCherry was incubated for
5 minutes in buffers with different pH-values. The remaining fluorescence intensity was measured (λEx=570 nm,
λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200. Furthermore, we normalized the fluorescence intensity at each pH value to the intensity at pH 7.
Detectable fluorescence can be measured in a pH-range from ph 4 to pH 12 while the pH-optimum is 6-7.
Interestingly, the fluorescence intensity seems to have a second optimum at pH 10-11. To verify this, we
measured the fluorescence-spectra of mCherry at pH 6 and pH 11 and compared them (Fig. 11).
Fluorescence spectra of mCherry at pH 6 (pink) and pH 11 (dark purple).
mCherry was incubated in buffers with different pH values for 5 min. The fluorescence spectrum of mCherry at pH 11
and pH 6 was measured (λEx=570 nm, gain calculated from 2.5 µM Texas Red) using
the TECAN infinite M200 and normalized to the maximum measured fluorescence.
The altered fluorescence spectrum of mCherry at pH 11 indicates, that the protein disintegrates at
higher pH which somehow results in increased fluorescence intensity. Maybe the altered protein structure
exposes the fluorophore to the media which minimizes inner protein quenching.
After expression, purification and characterization of mCherry we were able to define the optimal handling- and reaction
conditions.
Expression of mCherry using the IMPACTTM kit from NEB resulted in a higher yield but perhaps the cleaving
reaction should be performed at 4 °C
to avoid protein degradation. To maintain the full fluorescence intensity of mCherry one could consider decreasing
the amount of
DTT or using a different purification protocol, for example purification via his-tag.
An advantage of mCherry that we confirmed is the broad pH tolerance. Fluorescence is detectable from pH 4 to pH 12 while the pH optimum
is at pH 7. Further characterization additionally showed that mCherry should be kept in the dark as long as possible when handling it as
it shows photosensitivity like every other fluorescence protein.
