1. Development of new genetically encoded FRET probe
based on the fluorescent proteins Lumazine and Venus
MASTER IN BIOENGINEERING, Christophe Nell
In the laboratory of Professor Gerard Marriott, University of California Berkeley,
Supervised by Dr. Alexander Hoepker, UC Berkeley.
Under the direction of Prof. Philippe Renaud, EPFL
Materials and methods
Introduction
Results
Discussion
Reference Acknowledgements
The aim of this project is to study a new fluorescent recombinant protein composed of Lumazine protein (LumP) and Venus. The overlap spectrum between the
emission of LumP under excitation at 400nm and the absorption of Venus produce an efficient Förster Resonance Energy Transfer (FRET) phenomenon. I used this
system to develop new substrates for thrombin and MMP-2. Thrombin is a well know enzyme that allows us to characterize the efficiency of the transfer of energy
related to the cleavage[1]
. We know that the efficiency of the transfer (E) of energy is related to the distance R0
[2]
:
R0 can be experimentaly calculated with the overlap integral (J) between the emission of the donor LumP and the acceptor Venus.
n is the refractive indext of the middle, k² the orientation factor of the probe, ΦD the absolute quantum yield of the donor.
With the data recorded with LumP and Venus and the software flurotools[3]
, we obtain Ro = 52 angstroms. We recorded the change of emission spectrum and the
change of anisotropy during the cleavage of the substrates. The recombinant protein sensitive to MMP-2 was created because the level of this enzyme is a key
diagnosis for metastasis[4,5]
. Finally, the substrats were also used with a hydrogel to detect the activity of the enzyme with living cells using a confocal microscope.
3 differents substrats were produced to study this new recombinant protein: the first
without cleavage sequence and only 2 amino acids between the two proteins, the
second with 11 amino acids including -LVPR/GS- the sequence sensitive to thrombin,
and finally 13 amino acids including the MMP-2 substrate -IPVS/LRSG.
To record the change of emission spectrum, we used a PTI (Photon Technology
International) fluorometer and the software FelixGX 4.1.2. We recorded the change of
emission spectrum by looking at the emission intensity of LumP and Venus under
excitation at 400nm.
For the anisotropy, we used the luminescent spectrometer AB2 (AMINCO Bowman
Serie 2). The sample was excited and the emission intensity corresponding to the peak
of Lump was recorded with filters for the excitation and emission light. Then, the
anisotropy R can be calculated by the formula:
Finally, to record the cleavage of the probe with a hydrogel, we used a confocal
microscope.
3 different cell lines were cultivated on the new hydrogel: Hep-G2, NBT-2 and MDA-
MB-231. We did an overnight incubation before taking the images used to calculate the
amount of FRET on the surface of the gel.
On the left, the plasmid for the MMP-2
substrate. On the right, picture of the
bacteria BL21 expressing the
recombinant protein LumP-Venus taken
with the confocal microscope under
excitation at 405nm with a filter that
keeps only the light after 510nm.
Before the cleavage, the emission is maximal
at 530nm. After the cleavage, the emission is
maximal at 470nm, which is the peak of
emission of LumP.
V stands for Verical, the filter at 0° and H for a filter at 90°. The factor G
is a corrector factor for the instrument.
We have a 4.51-fold change for the ratio between the peak of emission of LumP and
Venus before and after the injection of thrombin. Similar results were recorded with the
MMP-2 sensitive substrate during the emission spectrum.
To track the cleavage of the recombinante proteins with the hydrogel, we illuminated it
with a 405nm laser. Then, we recorded the emission in two channels, before and after
510nm in order to separate the emission of LumP and Venus. To find out the activity of
MMP-2, we looked at the difference of ratio between the emission of LumP and Venus.
The graph on the left shows the different intensity and the ratio related to the yellow
line on the picture taken by the confocal microscope.
Emission spectrum recorded with
10μM of substrate and 14.7nM of
thrombin. The first recording at t0
is done without enzyme and the
curve in orange is done 18min
after the injection of the enzyme.
On the right, the ratios
470/530nm calculated from the
same experiment.
The anisotropy is calculated with the same
concentration than the emission spectrum. We
can see a decrease of the anisotropy from 0.245
to 0.166. It decreases because the cleavage
reduces the time between the absorption of the
photon and the emission of light. Moreover, a
smaller molecule tumble faster. This is why LumP
alone has a lower anisotropy.
On the left picture, MDA-MB-231 cells, objective
20x. The results of the Hep-G2 cells and the
NBT-2 cells are similar with no correlation
between a decrease of intensity in C2 related to
an increase of intensity in C1.
Because no real difference have been observed with the
cells, we produced a hydrogel with thrombin substrate
linked on the top of it. Then the enzyme was injected
directly into the buffer to mimick the action of MMP-2.
We can see that even if we loose emission intensity in
both channels, we have a significant change of ratio.
[1] Zhang, B., 2004. Design of FRET-based GFP probes for detection of protease inhibitors. Biochemical and Biophysical Research Communications,
323(2), pp.674–678.
[2] Valeur, B., 2001. Molecular Fluorescence: Principles and Applications Wiley-VCH. Available at: http://www.citeulike.org/group/2000/article/2395562
[3] a|e - UV-Vis-IR Spectral Software 1.2, FluorTools,, Available at: www.fluortools.com
[4] Yang, J. et al., 2007. Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor. Biochimica et biophysica acta,
1773(3), pp.400–7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17187878 [Accessed October 27, 2014].
[5] B. Packard, V. Artym et al., 2009. Direct visualization of protease activity on cells migrating in three-dimensions. Matrix Biol., 28(1), pp.3–10. Available
in PMC 2010 January 1.
[6] Piston, D.W. & Kremers, G.-J., 2007. Fluorescent protein FRET: the good, the bad and the ugly. Trends in biochemical sciences, 32(9), pp.407–14.
Available at: http://www.ncbi.nlm.nih.gov/pubmed/17764955 [Accessed July 11, 2014].
The recominant protein are linked to a 4-arm PEG SH/
Mal using MBS. Fibronectin with Traut's reagent is also
used to help the cells to stick on the gel.
We proved that the pair LumP-Venus separated by a protease specific sequence can be used for enzymatic study in the cuvette. However, the results with
the hydrogel did not allow to detect the protease activity of the cells. The lost of fluorescence on the gel during the cleavage by trombin in the two channels
shows that both, the stability of the protein and the attachement on the gel need to be improved. With the cells, no significant cleavage is detected as we
expect a change of ratio with an decrease of emission in the second channel C2 that is similar than for the emission scan with MMP-2 done in a cuvette.
Nevertheless, we can say that this new recombinant protein is suitable for enzymatic detection. The cleavage can be followed with different strategies and
this sustrates have different advantages compare to the probe currently used[6,4]
.
- The link between LumP and Venus can easily be changed and the recombinant proteins can be produced at low cost.
- The change of ratio between the peak of emission of LumP and Venus reaches 4.5-fold with 11 amino acids which is better than the usual pair CFP-YFP.
- The cleavage can easily be detected over time with an emission scan and the anisotropy of LumP.
- The change ratio for the emission intensity of LumP and Venus can be detected with a confocal microscope.
Emission scan before (t0)
and after the injection of
MMP-2
Prof. Gerard Marriott, who accepted me in his laboratory at UC Berkeley.
Dr. Alexander Hoepker, my supervisor during this project., who has taught me all the
techniques necessary to achieve this project. Moreover, the entire laboratory deserves a
special thank for the welcome and all the help that they personally provided me.
Prof. Heinis, who generously offered a stock solution of MMP-2 produced in his laboratory
of therapeutic proteins and peptides (LPPT) at EPFL.
Prof. Philippe Renaud, my supervisor from EPFL.
My family and my friends, old and new, who gave me support and made my stay a great
experience.
Cleavage of the substrats on
the hydrogel by thrombin