Development of Flexible Linker for Validation of
NanoLuciferase and mCherry BRET Assays
Calvin VanOpstall, PI: Brendan Looyenga, PhD, Calvin College
Introduction
v GluT1 is a passive glucose transporter for mammalian cells
which has been implicated in several diseases, including
Type I diabetes and several types of cancer. Understanding
what controls the activity of GluT1 may lead to possible
therapy targets for these diseases.
v The mechanism(s) by which GluT1 activity is controlled in
mammalian tissue are unclear, but may involve formation of
homodimers and homotetramers.
v Bioluminescent Resonance Energy Transfer (BRET) assays
allow for the measurement of protein-protein interactions
that occur within a specific distance known as the the
Förster Radius.
Preliminary Data:
Methods
Cloning Strategy:
Results
Cloning Results:
v Start from a 1x linker plasmid that contains the sequences for
both Nluc and mCherry on either side of a single 18 bp insert.
v Digest the plasmid with BamH1-HF and Xba1 restriction
enzymes to open the plasmid without losing the previous
insert.
v Based on the PCR screen of the clones, we are confident that
the variable length linker is indeed growing as expected and
will be useful as a control in the BRET assays.
v Our data support the preliminary BRET results that
suggested an GluT1 molecules begin to physically interact at
higher concentrations in the membrane; this is seen by
intermolecular BRET signal between GluT1 molecules.
v Ligate the digested plasmid with an annealed oligonucleotide
to increase the linker size by 6 amino acids (GGSGGS).
v The novel BRET pair of NanoLuciferase™ and mCherry are
working as expected and have proved to be a viable pair for
BRET assays
v Transform new extended plasmid into bacteria, isolate DNA,
and repeat.
v The linker series (1-10x repeats) that will be used to
calculate a Förster Radius for nLuc and mCherry has been
completed.
v Every insert adds a segment of known size to the peptide
linker that connects Nluc and mCherry, which allows a known
distance for calculating Förster Radii.
A
Conclusions
A PCR screen using primers flanking the variable region, which contains
the different length linkers, demonstrates a step-wise increase of 18 bp for
each step of oligonucleotide insertion into the parent plasmid.
BRET Results:
Future Directions
v BRET data from the linker series will be fit to a decay curve
to calculate the Förster Radius.
v Singly-labeled GluT1 fusion proteins are being used to
determine the actual distance between partners and to
determine the density of transporters required on the cell
surface to initiate multimerization.
B
Illustration of how the cloning strategy maintains each previous insert by destroying
the preceding BamH1 site, while at the same time creating a new BamH1 site at the
end of the insert for the next round of cloning. The Xba1 cut site remains the same
throughout, so the insert is always added to the correct position in the plasmid.
Aknowledgments
v Calvin College Department of Chemistry and Biochemistry
(A) A fusion protein in which mouse GluT1 was tagged with the
fluorescent protein mCherry and the bioluminescent protein
NanoLuciferase (nLuc) at either terminus can be expressed in cells.
Two types of reasonance energy transfer (RET) are possible for this
molecule: intramolecular BRET occurs between either terminus, where
as intermolecular BRET occurs between two separate molecules of the
fusion protein. (B) Expressing increasing amounts of the fusion protein
results in swtich from intra- to inter-molecular BRET signal.
v Chemistry Pfizer Fellowship
A cartoon illustration a completed variable length linker protein.
Objectives
v Develop multiple iterations of variable length linker that is
dual-linked with NanoLuciferase™ (nLuc) and mCherry
fluorescent protein.
v Determine the Förster Radii of nLuc and mCherry using the
linker series to compare to previously developed BRET
pairs.
v Use the linker series to determine whether the increase in
GluT1 BRET signal (Fig. 1) represents inter-molecular
association of GluT1 molecules.
v Dr. Brendan Looyenga
Variable linker Results of a BRET assay with the 1x-4x variable length linkers, as well as the duallinked GluT1, transfected at varying amounts of plasmid. There are three important
observations: 1) The BRET signal remains the same in all four linkers regardless of
the amount of DNA transfected; 2) The BRET signal in the linkers is decreasing as
the linkers get longer; and 3) BRET signal increases with increased plasmid amounts
for the GluT1 fusion protein, but is constant for the control linker proteins .
BRET Assay:
References
v Dacres, H.,Wang, J., Dumancic, M. M., & Trowell, S. C.
(2010, January 1). Experimental determination of the
förster distance for two commonly used bioluminescent
resonance energy transfer pairs. Analytical Chemistry,
82(1), 432-435.
v 293FT cells are plated at 10,000 cells/well of a 96-well plate
and allowed to adhere for ~24 hours.
v Cells are transfected with a 2-fold serial dilution of DNA
plasmid, from 50 ng to 6.25 ng per well.
v ~48 hours post-transfection, nLuc substrate (furimazine) is
added and fluorescence is immediately measured.
v The mean BRET ratio (610[LP]/410[80] nm) is calculated
for each well and normalized to the ratio of nLuc only.
v National Institutes of Health
Results of a BRET assay with the 1x-9x varialbe length linkers, as well as the duallinked GluT1, transfected at 50 ng of plasmid. The BRET signal from the 9x linker has
fallen below 50% of the signal from the 1x linker. This gives the ability to fit a decay
curve and calculate Förster Radius
v Drinovec, L., Kubale, V., Larsen, J. N., & Vreci, M. (2012,
August 28). Mathematical models for quantitative
assessment of bioluminescence resonance energy
transfer: application to seven transmembrane receptors
oligomerization. Frontiers in Endocrinology, 3(104),
1-10.