Expression and purification of hPIRT2 (TMEM100) end of semester research report

Remade for EDT310 Microsoft Word Project

Castro, M.

Student of Wade Van Horn

11/30/14

Abstract:
In the experiment conducted throughout the semester, the major goal was to express and purify hPIRT2 for
structural and functional analysis. Findings from the experiment include hPIRT2 expresses in BL21DE3 E. coli
and can be purified via Ni-NTA/Cation Exchange chromatography in 2-mg/L quantities. Purity and presence of
hPIRT2 was verified to be real by LC-MSMS and western blot/SDS-PAGE.

15

N hPIRT2 was also verified via

HSQC NMR analysis.

Introduction:
The transduction of
of the
unicellular
channels are a
high interest to the
Within the TRP
Venkatachalam,

are expressed
multitude of
sensations of cold
Daniel 2010;}} (see
Although
attention, there are
expressed from
phenotypes have
regulatory protein
TRPV1 {{36 Kim,
with heat and
channel {{34
2005;}}). PIRT is

signals across cell membranes is responsible for much

communication between cells in both multicellular and
organisms alike. Transient Receptor of Potential (TRP)
family of transmembrane proteins which have been of
biochemical/physical/pharmacological comminutes.
family are a series of sub-families {{38
Kartik 2007;}}{{37 Montell, Craig 2002;}} which
among a variety of life-forms and are involved in a
different signaling pathways, among them the
{{32 Peier, Andrea M 2002;}} and heat {{34 Vilceanu,
Figure 1).
transmembrane channels have received much
many other genotypes that have been found to be
from human DNA, however their corresponding
been relatively neglected. An example of this is a
named PIRT, which has been shown to modulate
Andrew Y 2008;}}, a TRP channel which interacts
capsaicin (and has been dubbed the heat sensative ion
Vilceanu, Daniel 2010;}} {{35 Zhang, Xuming
one of the better understood TRP modulators, but not
the only one.

Figure 1. TRP subfamily

homology {{38 Venkatachalam,
Kartik 2007;}}. There are 7
subfamilies of TRP channels,
expressed among metazoans.
Channels highly involved in
Figure 2. PIRT {{36 Kim, Andrew Y 2008;}} species sequence alignment. Human
cellular signal transduction.
PIRT shares high sequence alignment (in black) with PIRT from other species of
vertebrates. Expression of PIRT among these species suggests its importance for
many living organisms and allows the testing and comparison of PIRT from
different species

tentatively referred to as hPIRT2

Phosphoinositide Regulator of TRP
clear primary structure homology
and hPIRT has also been found to
vertebrates (see Figure 2). Because

Another good example of a

protein whose function and
structure have been pushed
aside is a two transmembrane
helix named TMEM100
which is conserved among
many terrestrial and
aquatic
vertebrates{{39

Figure 3. PIRT and PIRT2 sequence alignment.

Human PIRT and PIRT2 show high sequence
alignment. Similar transmembrane and binding
regions suggest that the structure-function
relationships could be related.

Somekawa,S. 2012;}}
(transmembrane
protein 100), which is
the protein of interest
in this paper. TEM100,
which will be
(human
Channels 2. It has very
with hPIRT (Figure 3),
have homology among
of the homology of
2

PIRT and PIRT2, the similarity of their functions in the body are inferred to be related, as well as their
importance in physiological functions.
In this study, hPIRT2 was expressed and purified in order to conduct in vitro studies on its interactions with
TRP channels and structural experiments using NMR.
Methods:
Expression
pDNA for hPIRT2 (in pET16b vector containing Amp resistance) was cloned in XL10 Gold E. coli and
miniprepped. Miniprepped pDNA was then transformed into BL121DE3, BL21DE3 Star and BL21DE3 Codon
Plus RP E. coli strains and expression tested in unlabeled M9 media at 18/25C at different concentrations of
IPTG (.33, .66 and 1mM). Culture samples were taken at T=0 (induction time), T=1 (12 hours after induction),
T=2 (24 hours after induction) and T=3 (36 hours after induction). The samples were frozen and ran on a Dot
Blot (western blot device).
The 14 darkest dots from the dot blot were then reran on a real western blot and coomasie SDS PAGE gel. The
darkest band from the coomasie/western was then selected for larger-scale expression of 15N hPIRT2. BL21DE3
E. coli were transformed only LB agar plate (containing ampicillin) and a colony was picked for each 3-mL
starter culture containing ampicillin. Starter cultures were grown at 37C for 8-10 hours and 1-mL of dense
starter culture was inoculated into a 1L flask of 15N M9 media containing ampicillin. Culture was rotated at
250rpm overnight (or 12 hours) at 25C to reach ideal induction OD of .6-.7. Culture was then induced with
1mM of IPTG for 24 hours at 25C. Culture was then harvested (OD usually around 1.2-1.5) using an
ultracentrifuge at 7000rpm at 4C. After centrifugation, supernatant from the centrifuge tube was discarded and
pellet was stored in a 50-mL falcon tube at -80cC until extraction.
Extraction
Cell pellet was retrieved from -80C freezer and thawed on ice. 25-mL of lysis buffer A (50mM Tris-HCl,
200mM NaCl, pH 7.2) was added to falcon tube containing pellet using a 25-mL Stripette. 250-L if PMSF was
then added the mixture, followed by 250-L of 1M CaCl2, 250-L of .5M Mg(OAc)2, and 50-L of LDR.
Mixture was solubilized using Stripette and left to tumble using a rotator at room temperature for 1 hour. The
mixture was then transferred to a metal beaker and sonicated at 4C for 15 minutes (50 amplitude, 5 seconds on,
5 seconds off). Empigen detergent was then added to the mixture to a final concentration of 3% (from 35%
stock) and left to tumble at room temperature for 1-2 hours. When solution became more transparent, the
mixture was then added into an ultracentrifuge tube and centrifuged at 18000rpm for 30-minutes at 4C.
Supernatant from tube was collected and the pellet was discarded. Supernatant was pH buffered back to 7.2 and
added to pre-equilibrated Ni-NTA resin. Ni-NTA + protein solution was tumbled at 4C for 1-2 hours. Mixture
was then gently centrifuged at 2000rpm and Ni-NTA was added into a chromatography column using a pipette.
Column was attached to UV-Vis spectrophotometer and chromatogram to prepare for purification.
Purification
Ni-NTA bound protein was washed in the column first using buffer A + 3% empigen (wash buffer 1). Once
chromatogram read no UV absorbance, buffer A + 1% empigen (wash buffer 2) was added at 5 CV (column
volumes). Then buffer A + .5% empigen (wash buffer 3) was added at 5CV. Next, buffer A with .5% empigen
and 20mM imidazole (wash buffer 4) was added at 5CV, eliciting an absorbance peak. Then, buffer A
containing .5% empigen and 40mM imidazole (wash buffer 5) was added at 5CV. Buffer A containing .5%
empigen and 60mM imidazole was then added at 5CV (wash buffer 6), and to complete the washing, buffer A, .
5% Empigen + 80mM imidazole was added at 5CV. The resin was buffer exchanged to Buffer A containing .5%
dodecylphosphocholine (DPC) at 5CV. Resin was eluted with 3CV of Elution Buffer (EB) containing buffer A, .
5% DPC, and 500mM imidazole. Elution fractions were quantified using spectrophotometer and stored at 4C.

Protein purity was determined using SDS-PAGE and western blot. If protein was impure, second round of NiNTA purification or cation exchange chromatography was used to gain >90% purity. For ion exchange, hPIRT2
elution sample was exchanged into a buffer containing .5% DPC, 50mM NaCl and pH 7.5 using a centrifuge
concentrator. Protein was added to equilibrated ion exchange FPLC column. Protein was washed and eluted
with a gradient increase of NaCl concentration (eluted around 500mM). Pure protein was collected and put into
NMR buffer containing Sample was concentrated down to 170-L (1mM hPIRT2) and added to a 5mm
NMR tube with 4-L of D2O. Tube was inserted into an 850MHz NMR magnet and a HSQC experiment was
ran.
Results and Discussion:

Figure 4. Dot blot. Qualitative expression test of

hPIRT2 shows protein as dark dots. 3 cell lines/2
temperatures (columns) and 3 IPTG
concentrations/negative controls (rows).

Figure 5. Coomasie of hPIRT2. SDS-PAGE of dot blot

samples, showing a dark hPIRT2 at 15kDa region.

14

25 kDa
20 kDa

Figure 7. Western blot. Messy hPIRT2 showing up at 15kDa region, verifying success of Ni-NTA purification. Sample
wash smeared due to high detergent concentration (3% Empigen).

hPIRT dot blot demonstrated that the expression of hPIRT2 was very strong, due to detection by antiHis
antibody (Figure 4) . The dot blot is organized in such a way that the concentrations of IPTG (in mM) in rows A
and E are .33, B and F are .66, C and G are 1, and D and H represent the negative control groups (no induction).
Columns 1, 2 and 3 represent cell lines BL21DE3, BL21DE3 Star and BL21DE3 Codon Plus RP at 18C.
Columns 4, 5 and 6 are the same respective cell lines tested at 25C. It is very clear that there is nonspecific
binding by the smearing present in the control rows. The presence of hPIRT2 was inconclusive, so samples were
reran on a coomasie/western blot to compare versus size. It was very clear from the coomasie that there was
hPIRT2 by a dark band at the 15kDa region of the gel. Because of the 6xHistadine tag, the proteins weight was
brought much closer to 15kDa from 14.3kDa. All of the bands looked dark, and indistinguishably so from one
another. For that reason, a shotgun approach was used
and BL21DE3 with 1mM IPTG at 25C was used for the
15
N hPIRT2 expression for analysis.

Figure 6. Ni-NTA purification chromatogram. From

right to left, peaks read: flowthrough, wash1, wash2,
wash3, wash4 and elution1.

The purification of 15N hPIRT2 yielded a large amount of

sample in the elution fraction, as seen in Figure 6. From
right to left, the chromatogram reads FT (flow through),
W1-W4 (Wash 1-4, skipping the DPC exchange steps),
and E1 (Elution). The elution peak is much larger than
any of the previous wash peaks, which likely were
knocking off nonspecific proteins from the Ni-NTA
resin. Figure 6 shows a chromatogram for an elution
fraction that was eluted in 3% Empigen. The high
concentration of Empigen caused a smudging in the
western blot analysis. In Figure 7, a prominent dark
smearing region directly through the 15 kDa area can be
seen, however, lots of nonspecific binding or smearing
also appeared to be present at larger molecular weights.
It was clear that the protein wasnt pure, but there was a
lot of it. At this point, a further purification was needed
to get rid of the nonspecific proteins. The cation
exchange column proved to be a useful chromatographic
tool for purifying hPIRT2 from its impurities. The
estimated pI of hPIRT2 is approximately 9.5, so a cation
5

exchange solution with the pH of 7.5 was used to ionize it. A FPLC was used to automate the process, which
used a gradient elution to knock off the protein. As seen in Figure 8, the protein is begins eluting at mM
NaCl, and impurities begin to follow it at higher NaCl concentrations. To ensure purity of the samples, the
FPLC automatically fractionated samples into .5-mL vials. The vials containing high concentrations of hPIRT2
were combined (all samples had few amounts of impurities, about <10%) and quantified, yielding
approximately 2-mg of pure protein per liter of M9 culture. The purity of the samples was tested via SDS-PAGE
(Figure 9), and the pure hPIRT sample was sent off for LC-MSMS for verification. 14N hPIRT was used for LCMSMS because of the price of labeling proteins with 15N. The pure samples were then swapped into NMR
25 kDa
buffer
containing DPC to begin detergent screening via NMR (Figure 10).
20 kDa

Figure 9. SDS-PAGE of hPIRT2. Purified (after second Ni-NITA run) hPIRT2 was verified using 15% acrylamide gel.
Band at 15kDa was cut out and sent for LC-MSMS analysis.

HSQC experiment of 15N hPIRT2 was done

two temperatures. 40C, as shown in red,
25C in black. The downfield shift with
increasing temperature is likely due to the
increased kinetics of the molecules,
revealing more peaks as tumbling increases.
peak corresponds to an amide 15N-1H bond.
Micelles in sample are likely concealing
many of the membrane region peaks,
implying that either DPC isnt the best
detergent, or that micelles in general are not
best membrane mimic for NMR studies on
protein.

at
and

Each

the
this

Conclusion:
The goal of the semester was to fully express
purify the protein to an extent that NMR
studies would be applicable. The major
conclusion of the project so far is that this
the first successful recorded full expression
purification of hPIRT2 that actually made it
NMR magnet. Although the results are

and
was
and
to an
Figure 10. HSCQ spectrum of hPIRT2. NMR spectrum
showing amide backbone peaks of hPIRT2. In black,
temperature of 25C shifts signals up-field. In red,
temperature of 40C causes downfield shift, also exposing
more peaks.

preliminary, the future of this project is very bright. Future directions of the project include detergent micelle
screening, insertion of hPIRT2 into nanodiscs, in vitro interactions of hTRPM8-VSD and hTRPV1-VSD by
NMR and pull-down analysis. The data from these studies will be used to advance the biophysical and structural
understanding of ion channels and their interactions with modulator proteins.