Matthew King

BSCI 399
IBBR Xiao Lab
Abstract
My work within the Xiao Lab has been to determine the optimal procedure to purify the
Arabidopsis thaliana protein RPW8.2 from E. coli in a quantity substantial enough for X-ray
crystallography, NMR and production of polyclonal antibodies. A range of 20mg 80mg per liter
of media is desired. Optimization of RPW8.2 expression is currently being investigated by
controlling conditions like induction time, concentration, temperature, and media while
purification of RPW8.2 is being investigated by testing several HIC columns. The optimal
method thus far is to induce E. coli to express RPW8.2 with a GST-Halo tag and purify through a
GST column. This method has given the largest isolated bands, however at this stage quantitative
work is not feasible.
Introduction
Sixteen to eighteen percent of global crop failure is due to pathogens. Powdery mildew is
one of the most common diseases of crops caused by Ascomycete fungi in the order of
Erysiphales (Gllner). Isolates of the Golovinomyces cichoracearum species are capable of
causing powdery mildew diseases in hundreds of dicot plant species. Powdery mildew infects
plants by releasing spores, dispersed by wind, water or insects, onto uninfected leaves (Braun).
When these spores become hydrated they germinate and grow a germ tube that swells into a
structure called an appressorium due to physical and/or chemical queues (Braun).

Figure 1: Drawing showing the plasma membrane of a plant cell with an appressorium,
haustorium, extrahaustorial matrix, extrahaustorial membrane and RPW8 proteins being
secreted from the Endoplasmic reticulum.

Figure 2: RPW8 locus tagged with the sequence for the yellow fluorescent protein (YFP).
The picture on the left shows the appressorium punctured through the plasma membrane
of the Arabidopsis thaliana cell with a haustorium that has RPW8-YFP proteins within its
extrahaustorial membrane. The proteins are also dispersed within the cytosol.

The appressorium uses turgor pressure to penetrate the cell wall and invaginate the
plasma membrane of the host cell to gain access to the nutrients of the cytoplasm (Braun). This
bulging feeding structure is called the haustorium and is considered the host-pathogen interface
in this particular infection (RPW8.2 Targeting 2009). The haustorium also functions to secrete
effector proteins into the host cell across the interface to suppress host defenses (RPW8.2
Targeting 2009). Due to selective pressures imposed by pathogens, plants have evolved post
invasion resistance mechanisms often controlled by dominant resistance (R) genes that directly
or indirectly detect specific pathogen effectors and trigger effective defense responses (RPW8.2
Targeting 2009). One gene, RPW8.2 confers broad spectrum resistance to fungi under the genus
Golovinomyces as opposed to resistance to specific species of fungus as do many other R genes
that confer resistance (RPW8.2 Targeting 2009). RPW8.2 is specifically targeted to and interacts
with the extrahaustorial membrane (RPW8.2 Targeting 2009).
Despite much of the genetic discoveries and insights into mechanisms using fluorescent
microscopy the RPW8.2 structure has not been solved. Protein structure elucidates function, and
a structure will greatly aid in understanding the functional components of RPW8.2 and its
connection to powdery mildew resistance. The RPW8.2 protein has only been purified to the
point that it can be cut from an SDS PAGE gel and sequenced. To be able to determine the
secondary and tertiary structure using X-ray crystallography as well as NMR a substantial
amount of the RPW8.2 protein would have to be purified. Developing a way to purify RPW8.2
has been my main focus in the Xiao Lab involving induction of E. coli and many trials of
supernatant purification through a variety of columns.
Research of the purification of RPW8.2 involves investigation at the cellular level and at
the molecular level. Investigation at the cellular level research involves cell culture,

transformation, induction and model organisms. Investigation at the molecular level research
involves exploiting the known properties as well as tagging the RPW8.2 protein so that it can be
isolated using column chromatography.

Figure 3: A drawing of RPW8.2 tagged with GST and Halo in tandem.

Materials and Methods

RPW8.2 is 174 amino acids. Unlike most proteins which have a slightly acidic isoelectric
point (pI), RPW8.2 has a pI at pH 9.3. Additionally, a transmembrane domain and a high
percentage of hydrophobic residues comprising about 34% of all amino acids in RPW8.2 make
this protein difficult to express in E. coli. A soluble epitope tag (GST) was added to RPW8.2 and
resulted in a slight increase in expression. Halotag, a new protein epitope from Promega has also
been fused upstream of RPW8.2 in-tandem translated with GST. The GST tagged proteins must
be expressed using the GE Healthcare pGEX series of expression vectors. This dual-epitope
strategy further increased the proteins expression.
Figure 3: pGEX-4T-1 plasmid used to transform the RosettaTM pLyseS BL21(DE3)
competent cells.

Cell Culture
RosettaTM pLyseS BL21(DE3) competent cells transformed with the plasmid pGEX-4T-1
was used from stock cell pellets from a -80 C freezer. The pGEX-4T-1 plasmid contained genes
for ampicillin as well as chloroamphenicol resistance in addition to the RPW8.2 tagged with dual
epitopes, GST and Halo proteins. The cells were then deposited onto a sterile pipet tip and
dropped into an Erlenmeyer flask in (50mls) of Lysogeny Broth (LB) media containing 100 g
per ml of Ampicillin and 50 g per ml of Chloroamphenicol. This culture is then incubated in a
37 C shaker overnight. 1 ml of this culture is added to a 1L solution of LB media. This 1L broth
is then placed in a 30 C shaking incubator. Absorbance of small 1ml samples are taken about

every hour and then more rapidly as the Absorbance reached the desired OD of 0.8. When the
culture reached an OD of 0.8, 1mL of Isopropyl -D-1-thiogalactopyranoside (IPTG) is added to
the 1L of culture to induce E. coli to express the proteins in the Lactose operon including GSTHalo-RPW8.2 which resides in the lactose operon. The culture is then incubated for an additional
two hours. After two hours the culture is decanted into two 500mL centrifuge tubes and spun at
4500g for 20 minutes. The supernatant was poured out and then the cells were re-suspended in
50mL of (20mM Phosphate buffer). The re-suspended cells are then placed on ice so that they
can be lysed in a sonicator for 4 min with a 10 sec on and 10 sec off interval. This lysate was
then aliquoted into two 30 mL centrifuge tubes and spun at 10,000g for 20 min at 4 C. The
supernatant was then filtered through a 45 m filter using a luer-lok syringe system and is ready
to be loaded onto a Column.
GST Column
The first column always used is the GSTrap FF 1ml column from GE Healthcare to purify
glutathione S-transferase tagged proteins. The binding buffer is phosphate buffer saline (PBS)
which is composed of 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 at pH
7.3. The elution buffer is composed of 50 mM Tris-HCl 10 mM reduced glutathione at pH 8.0.
All fluid was loaded onto the column using a 10 ml luer-lok syringe. The first step was to
equilibrate the column using 5 mls of the binding buffer PBS. After equilibration the filtered
lysate is then applied to the column at a flow rate of 1 ml/min. After all 50 ml of filtered lysate is
pushed through the column, the column is then washed with 5 ml of PBS binding buffer at a flow
rate of 2 ml/min. After washing, 10 mls of glutathione elution buffer is applied to the column at a
flow rate of 1 ml/min to elute GST-Halo-RPW8.2. Flow-through from the loading step as well as
the elution step were collected. The flow through from the elution step was split into two

centrifuge concentrator tubes and spun for at 4500g for 20 min. The concentrated protein is then
incubated in a 37 C incubator overnight with Tobacco Etch Virus Protease (TEV protease) to
cleave the GST-Halo tag from RPW8.2. The resulting product is then stored in the -20 C freezer
in a microfuge tube.
Cation Exchange Column
A HiTrap SP FF 1 ml column from GE healthcare is used to purify the cleaved RPW8.2 based
upon the high pI of pH 9.3. The binding buffer is 20 mM BICINE buffered to pH 8.4 using
NaOH. A stepwise gradient of NaCl was used to elute the protein. The concentrations of NaCl
were all made in a standard concentration of 20 mM BICINE at pH 8.4. The concentration of
NaCl were 0 mM, 500 mM and 1M. To prepare the cut RPW8.2 for loading onto the column, 1
ml of the concentrated protein was diluted into 10 ml of binding buffer. All fluid was loaded onto
the column using a 10 ml luer-lok syringe. The column is first equilibrated with 5 mls of 20 mM
BICINE binding buffer. The 11 ml of protein solution was then loaded onto the column at a flow
rate of 1 ml/min. The column is then washed with 5 mls of 20 mM BICINE binding buffer at a
flow rate of 2 ml/min. After washing, the stepwise gradient was pushed through the column. 5
mls of 0 mM NaCl, 500 mM NaCl and 1M NaCl was pushed through the column with a flow
rate of 1 ml/min. The flow through was collected from the loading step as well as the elution of
the stepwise gradient. The 5 mls for each NaCl concentration was decanted into centrifuge
concentrator tubes and spun for at 4500g for 20 min. The resulting product is then stored in the
-20 C freezer in a microfuge tube.
Butyl Column

A HiTrap Butyl FF 1 ml column from GE Healthcare was used to purify RPW8.2 due to 30% of
the protein containing hydrophobic residues. The binding buffer used was 1 M ammonium
sulfate (NH4)2SO4 and 50 mM sodium phosphate at pH 7.0. A stepwise gradient of ammonium
sulfate was used to elute the protein. The concentrations of ammonium sulfate were all made in a
standard concentration of 50 mM sodium phosphate at pH 7.0. The concentrations of ammonium
sulfate were 1 M, 0.5 M, 0.35 M, 0.25 M, 0.15 M and 0M. To prepare the cut RPW8.2 for
loading onto the column, 1 ml of the concentrated protein was diluted into 10 ml of binding
buffer. All fluid was loaded onto the column using a 10 ml luer-lok syringe. The column is first
equilibrated with 5 mls of 1M ammonium sulfate binding buffer. The 11 mls of protein solution
was then loaded onto the column at a flow rate of 1 ml/min. The column is then washed with 5
mls of 1 M ammonium sulfate binding buffer at a flow rate of 2 mls/min. After washing, the
stepwise gradient was pushed through the column. 5 mls of 1 M, 0.5 M, 0.35 M, 0.25 M, 0.15 M
and 0M ammonium sulfate was pushed through the column with a flow rate of 1 ml/min. The
flow through and the elution of the stepwise gradient was collected from the loading step. The 5
mls for each step was decanted into centrifuge concentrator tubes and spun at 4500g for 20 min.
The resulting product is then stored in the -20 C freezer in a microfuge tube.
Phenyl Column (AKTA FPLC)
HiPrep 16/10 Phenyl FF 20 ml column from GE Healthcare was used to purify RPW8.2 due to
30% of the protein containing hydrophobic residues. The binding buffer used was 1 M
ammonium sulfate (NH4)2SO4 and 50 mM sodium phosphate at pH 7.0. A linear gradient of
ammonium sulfate was applied to elute the protein using an AKTA FPLC machine operated
through UNICORN software. To prepare the cut RPW8.2 for loading onto the column, 1 ml of
the concentrated protein was diluted into 10 ml of binding buffer. The column is first equilibrated

with 100 mls of 1M ammonium sulfate binding buffer. The 11 mls of protein solution was then
loaded onto the column at a flow rate of 1 ml/min using a luer-lok syringe. The column is then
washed with 100 mls of 1 M ammonium sulfate binding buffer at a flow rate of 2 mls/min. After
washing, 50 ml of 1 M ammonium sulfate was applied to the column before beginning the linear
gradient which was applied to the column at a rate of 2 mls/min with a slope of 0.4% increase in
50 mM sodium phosphate per every ml. The elution was collected in 5 ml fractions. The 5 ml
fractions were decanted into centrifuge concentrator tubes and spun at 4500g for 20 min. The
resulting product is then stored in the -20 C freezer in a microfuge tube.
Results

Figure 4: SDS PAGE Gel analyzing the elution from a GSTrap FF 1ml column. Shows all
the bands very clearly and is the optimal way to purify RPW8.2 thus far. The Gel shown
here was ~15% acrylamide.

Figure 5: SDS PAGE Gel analyzing the elution from a HiTrap SP FF 1 ml column. Most of
the protein came out with the 0M NaCl. There is some residual GST-Halo from the 500mM
NaCl. RPW8.2 did not bind to the column and was released during the first step of the
gradient. This gel is ~10% acrylamide.

Figure 6: SDS PAGE Gel analyzing the elution from a HiTrap Butyl FF 1 ml column.
RPW8.2 did not bind to the column and must have eluted in the washing step. GST-Halo
and GST are the only proteins that are shown to bind to the column. RPW8.2 was not
present in the first step of the gradient and so it did not bind to the column.
Figure 7: This is a graph from the UNICORN software used to run the AKTA FPLC
machine. The blue, brown and green lines correspond respectively to UV absorbance at
280nm, conductance in Siemens and the theoretical linear gradient slope. The peak shown
in both the blue and brown lines represent the eluted protein. The peak occurs in fractions
1 through 10 when 50 mls of 1 M ammonium sulfate was being applied. It should also be
noted that the plateau after the peak of the blue line is due to a switch from UV at 260nm to
UV at 280nm, this is a mistake that was fixed during the procedure and can be visualized in
the graph.

Figure 8: SDS PAGE Gel analyzing the elution from a HiPrep 16/10 Phenyl FF 20 ml
column. Fractions 4-9 were used to run on the gel because they corresponded to the largest
part of the UV absorbance peak. The fractions showed little protein and there was no
difference between the fractions. Lanes 1-3 were not used because the acrylamide gel was
warped in that area.
Discussion
This work towards determining the optimal procedure to purify the Arabidopsis thaliana
protein RPW8.2 from E. coli is the farthest anyone has taken this project due to my initiative to

make this my main goal. Many members of the Xiao lab have tried to purify RPW8.2 as well as
other RPW8 proteins without progress. Most members are doctoral candidates or post doctorates
who dont have the time or funding to waste on an elusive goal such as RPW8.2 purification. I
have been able to implement other members ideas as well as my own to develop experiments
and have even applied for a grant from HHMI to create support for this project.
Funding is needed for the continuation of this project because all of the viable columns in
the Xiao lab have been tested and shown above, however, the Xiao lab does not have the
finances to buy new columns. Despite this hurdle, the information gained from doing these
experiments has given a lot of insight into what can be done currently with the protein as well as
what is to be done in the future. This information has reinforced many of the already known
procedures such as the GSTrap FF column. Also the use of 15% acrylamide instead of 10%
acrylamide is much better for visualization of RPW8.2 due to its small size easily running off the
gel. The HiTrap SP FF column shows some promise due to the fact that RPW8.2 was present in
the first step of the gradient although it did not have good separation from the other proteins.
Both the HiTrap Butyl FF and HiPrep 16/10 Phenyl FF do not show much promise because
RPW8.2 was not shown in the SDS PAGE acrylamide gels. Despite the negative results from the
columns, the variety of columns tested as well as the use of a linear gradient from the AKTA
FPLC is a strength of this work. However, after showing that one column could be tested using
AKTA FPLC machine, that fact that all of the columns could have been tested using the AKTA
FPLC appears as a weaknesses of this work. A variety of buffers could have been tested to make
certain that the column of interest was not a viable option for RPW8.2 purification.
Future research would involve doing a linear gradient with the AKTA FPLC
machine for the HiTrap SP FF column and the HiTrap Butyl FF column. Also testing other

binding and elution buffers to see if alternative salts make a difference. Using other columns
such as HisTrap for Histidine tagged RPW8.2 is possible. Also size exclusion preferably using a
column or possibly using gel electrophoresis with agarose is a method that may have been
simply overlooked. If enough protein from a GSTrap FF column was run on an agarose gel
multiple times it might be enough to send away for production of polyclonal antibodies. These
antibodies could then be used for isolation of a large amount of the protein. Finally the use of a
different organism to express the protein could be used, one such organism is the methylotrophic
yeast Pichia pastoris. Yeast are eukaryotic and therefore have an ER and Golgi apparatus unlike
E. coli which could provide the same post-translational modifications as well as codon bias
available in plants. This could not only yield a RPW8.2 protein with greater similarity to the
RPW8.2 protein in Arabidopsis thaliana but also yield a protein with stronger properties making
it easier to isolate. Thus far the two experiments that are currently being planned are using a
HisTrap for Histidine tagged RPW8.2 and using transformed Pichia pastoris to express RPW8.2.
Bibliography
Braun EJ, Howard RJ. (1994). "Adhesion of fungal spores and germlings to host-plant
surfaces". Protoplasma 181 (14): 20212. doi:10.1007/BF01666396
Gllner K, Schweizer P, Bai Y, Panstruga R. Natural genetic resources of Arabidopsis thaliana
reveal a high prevalence and unexpected phenotypic plasticity of RPW8-mediated
powdery mildew resistance. New Phytol. 2008;177(3):72542. Available at:
http://www.ncbi.nlm.nih.gov/pubmed/18211475. Accessed November 18, 2010.
Wenming Wang, Yingqiang Wen, Robert Berkey and Shunyuan Xiao. Specificc Targeting of
the Arabidopsis Resistance Protein RPW8.2 to the Interfacial Membrane Encasing the

Fungal Haustoriium Renders Broad-Spectrum Resistance to Powdery Mildew. Plant

Cell 2009 21: 22898-2913. First Published on September 11, 2009;
doi:10.1105/tpc.109.067587