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).
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).
To further characterize mCherry in vitro, we purified mCherry comparing two different purification
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).
The expression culture of mCherry in pTXB1 showed a brighter red color which indicates a higher expression level.
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).
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
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).
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
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.
To gain some more knowledge about mCherry we analyzed different properties of the protein. First, we measured its
emission- and absorbance spectra (Fig. 7).
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).
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).
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).
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).
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
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.