Biochemistry 528 Paper 11

Introduction
Eukaryotic cell division requires intimate spatial and temporal coordination of cell growth,
chromosome replication, segregation, and cytokinesis (Luka and Winey, 1998). The cell cycle
consists of two stages: interphase and M phase, where nuclear division (mitosis), and
cytoplasmic division (cytokinesis) make up the M phase, and the Gap-1 phase (G1), S phase
(DNA replication) and Gap-2 phase (G2) make up interphase. In mitosis, the cell goes through
prophase, prometaphase, metaphase, anaphase, and telophase. Once mitosis is completed,
daughter cells begin a new interphase in which the cell is prepared for proper division. Each
phase of the cycle is regulated in a cyclin-Cdk (cyclin-dependent kinase) dependent manner,
where Cdks are constitutively expressed, and cyclin molecules are only synthesized in specific
phases. The entry into mitosis is initiated by the activation of cyclin-Cdk complexes, leading to
chromosome condensation, mitotic spindle formation, and various metaphase events (Tth et al.,
2007). Cohesin complexes aid in holding replicated chromosomes together and once metaphase
is completed, separase (a protease) is activated and cleaves a subunit of the complex to allow
splitting, and separation of sister chromatids in anaphase (Tth et al., 2007). The activation of
separase involves the anaphase promoting complex (APC) and its co-activator, Cdc20. Together,
they form an ubiquitin ligase that rapidly degrades securin (separase inhibitor), as well as Clb2,
which leads to a reduction in Cdk activity (Tth et al., 2007). The completion of mitosis is
marked by a reduction in Cdk activity and is required for the exit of the mitotic phase. The
ubiquitin ligase, however, does not allow a large enough reduction of Cdk activity to allow
complete exit of mitosis (Tth et al., 2007). Even a reduction in Cdk activity alone is inadequate
for mitotic exit, as phosphorylated cyclin-Cdk complexes need to be dephosphorylated to
become inactive (Tth et al., 2007). Thus, an alternate system must exist for complete mitotic
exit.
In Saccharomyces cerevisiae, the mitotic exit network (MEN) acts as a signaling pathway
that administrate events related to mitotic exit (Stoepel et al., 2005). These events include the
inactivation of Cdks, promotion of cytokinesis, activation of G1 gene transcription, and
formation of pre-replication complexes (Stoepel et al., 2005). Regulatory proteins involved in
this network include: protein kinases (Cdc5p, Cdc15p, Dbf2p, and Dbf20p), a phosphatase
(Cdc14p), a GTPase (Tem1p), a GEF (Lte1p), and Mob1p (Stoepel et al., 2005; Weiss et al.,
2002). Mobs (Mps one binding) are a unique family of non-catalytic proteins that are highly
conserved in all eukaryotes. In yeast, Mobs interact with Dbf2-like proteins, a subfamily of
serine/threonine kinases (Mrkobrada et al., 2006). The Mob-Dbf2 complex is reminiscent of how
the cyclin-Cdk complex works (Mrkobrada et al., 2006). Tem1p is regulated by Lte1p, and a
two component GAP, Bub2p-Bfa1p (Stoepel et al., 2005). In late anaphase, Cdc5p inhibits
Bub2p-Bfa1p, and leads to the accumulation of GTP-Tem1p, activating Cdc15, which
phosphorylates and activates Mob1p-Dbf2 (Stoepel et al., 2005). Active Mob1p-Dbf2p indirectly
causes the release of Cdc14p, a proline directed phosphatase that drives mitotic exit through
dephosphorylation, and inactivation of Cdks (Stoepel et al., 2005; Weiss et al., 2002). Until
anaphase is reached, Cdc14p remains inhibited by Net1 in the nucleolus. When signaled to be
released, Cdc14p does so in two regulatory pathways (Stoepel et al., 2005). The initial phase of
release occurs at early anaphase via FEAR (Cdc fourteen early anaphase release) proteins (polo-
like kinase cdc5p, separase Esp1p, and kinetochore protein Slk19p) (Stoepel et al., 2005). The
second phase of release occurs at telophase, and is dependent on MEN proteins (Stoepel et al.,
2005). Once released, Cdc14p targets late mitotic substrates, such as Cdh1p, sic1p and Swi5p;
Cdk substrates that regulate mitotic cyclin degradation, Cdk inactivation, and G1 gene
transcription, respectively (Yoshida and Toh-e, 2001).
In S. cerevisiae, Mob2 exists as a Mob1-like component of the Ram (regulation of Ace2p
activity and cellular morphogenesis) signaling network and interacts with Cbk1, a
serine/threonine kinase similar to Dbf2. Mob2 activates Ace2p, a transcription factor which
direct daughter cell-specific transcription of genes required for septum degradation and cell
separation (Hou et al., 2003; Weiss et al., 2002). Thus, Mob2p-Cbk1p works analogously to
Mob1p-Dbf2p in MEN (Weiss et al., 2002).
The septation initiation network (SIN) in fission yeast (Schizosaccharomyces pombe) is
equivalent to MEN, but is only involved in cytokinesis, and Cdk activity reduction (Citterio et
al., 2006). The network involves protein kinases (Cdc7p, Sid1p, Sid2p. and Plo1p), a GTPase
(Spg1p), a GTPase-activating protein complex (Cdc16p-Byr4p), a SPB anchoring complex
(Cdc11p-Sid4p), and a Sid2p binding protein Mob1p (Salimova et al., 2000; Simanis, 2003). The
activation of Mob1p-Sid2p via Cdc7p, and sid1p-cdc14p allow localization of Mob1p-Sid2p to
the contractile ring, promoting septum formation events (Yoshida and Toh-e, 2001).
Some constituents of the MEN and SIN are well conserved from yeast to higher
eukaryotes. In humans, six Mob proteins have been identified: HsMob1A, HsMob1B, HsMob-
LakA, HsMob-LakB, HsPhocein, and HsMob2 (Wang, 2006). While more extensively studied in
yeast, the functions of MEN/SIN related networks have not been resolved in higher eukaryotes.
For example, although recent studies have shown that HsMob1 and HsMob2 interact with Ndr1
and Ndr2 kinases, the functions of these interactions are unclear (Mrkobrada et al., 2006).
Furthermore, other possible Mob-interacting proteins in higher eukaryotics have yet to be
identified. In S. cerevisiae, proteins which interact with Mob1 and Mob2 have been found
through a variety of methods including: two-hybrid, affinity capture, co-crystallization, and
biochemical activity assays. In addition, the Saccharomyces Genome Database (SGD) was
recently developed to compile the molecular biology and genetics of budding yeast, showing all
Mob-interacting proteins. No such database has been built for higher eukaryotes. Consequently,
the goal of this study is to amplify our knowledge of Mob and Mob-interacting proteins in higher
eukaryotes, specifically in Xenopus laevis, using human Mob proteins as the bait in pull down
assays. Since Mob protein interactions have been found to regulate the cell cycle in yeast, and
are conserved in higher eukaryotes, it is possible that analogous interactions play significant
roles in higher eukaryotic cell cycles. Thus, the search and identification of Mob-interacting
proteins in this study will allow future characterization of the nature of these interactions.
To identify potential Mob-interacting proteins, bait and prey pull down assays were
employed. An E.coli expression system was used to over-express the bait proteins which
included HsMobLak, HsPhocein and HsMob2. Each bait protein was expressed as glutathione-S-
transferase (GST) or histidine tagged fusion proteins, purified using French Press and column
affinity chromatography. With these columns of bait protein, Mob-interacting proteins contained
in Xenopus laevis egg extracts were captured and eluted. Samples were concentrated using
Bradford assays and Millipore membrane filters. SDS-PAGE was useful in detecting possible
Mob-interacting protein bands and mass spectrometry provided a method to decipher the
composition of these bands. Moreover, frog eggs are large, with an abundant storage of proteins
required for rapid cell cycles that follow fertilization. Finally, information obtained about frogs
can be applied to other similar vertebrates, as well as humans.
Material and Materials
Over-expression of Mob Bait Proteins
A. GST-HsPhocein(XM-03)
A BL21 (DE3) E.coli strain harbouring a pGEX-4T1-full length XM-03 construct was
streaked onto a Luria Bertani (LB) plate containing 75ug/ml ampicillin (company name) and was
subsequently incubated at 37oC for twenty-four hours. Following, a single colony was inoculated
into fresh enriched LB medium (50ml) containing 75ug/ml ampicillin. The culture was incubated
in a shaker (type of shaker) for twenty-four hours at 37oC and 200 rpm. Next, a dilution of the
culture (1:50) was made into 450ml of fresh enriched medium containing 75ug/ml ampicillin.
Incubation of the diluted culture in a shaker at room temperature at 200 rpm was then conducted
until the optical density reading at 600nm was between the values, 1.0 to 1.5. At this point,
protein expression was induced by the addition of 0.4mM IPTG (company name) for five hours
with continual shaking at 200 rpm and incubation at room temperature. Cell pellets were
harvested via centrifugation at 6000 rpm (Beckmen JLA-16.250 rotor) at 4oC for fifteen minutes
and stored at -80oC until purification.
B. GST-HsMob2
An E.coli BL21 (DE3) strain harbouring pGEX-5T1-full length HsMob2 construct (Wang,
unpublished) was grown with the same procedure and conditions as those employed for HsMob-
Lak expression. However, 75ug/ml ampicillin was used in replace of 30ug/ml kanamycin.
C. Glutathione-S-Transferase Control
The GST-control was used for experiments involving GST-tagged proteins in this study. An
E.coli DH5- strain (Wang, unpublished) harbouring the GST-tag with no protein insertion was
streaked onto a LB plate containing 75ug/ml ampicillin. Inoculation, dilutions, expression,
harvesting and storage procedures are identical to those used in the expression of GST-
HsPhocein.
D. 6xHis-HsMob-Lak (XM-05)
An E.coli BL21 (DE3) strain harbouring the pET29a-full length XM-05 construct (Wang,
unpublished) was grown on a LB plate containing 30ug/ml kanamycin, (company name
) at 37oC for twenty-four hours. A single colony was inoculated into 2ml of fresh enriched LB
medium containing 30ug/ml kanamycin. The culture was incubated in a shaker at 37oC and 200
rpm for twenty-four hours before being diluted into 50ml of fresh medium, containing 30ug/ml
kanamycin. The diluted culture was then incubated in a shaker for an additional twenty-four
hours at 37oC and 200 rpm. The culture was diluted once again (1:100), into fresh enriched LB
medium with 30ug/ml kanamycin, and subsequently left to grow at room temperature with
continual shaking, until the optical density at 600nm was 1.0 to 1.5. The method for protein
expression, harvesting of cells, and storing of pellets was identical to that of HsPhocein.
E. Ni-NTA-6xHis-B2Nt-control
The Ni-NTA column, saturated with 6xHis-B2Nt was used as a control for experiments
involving proteins tagged with six tandem histidines. The E.coli strain harbouring the construct
containing B2Nt was grown on a 75ug/ml ampicillin LB plate. Several cultures were inoculated
into 100 ml of enriched media, and left to grow for twenty-four hours at room temperature with
continual shaking (300 rpm). 100ml of fresh enriched media was then added to the culture in
addition to 0.8mM IPTG. Subsequently, the culture was permitted to grow for four hours at
37oC. Cells were then harvested by centrifugation at 4000xg for five minutes (Beckman JS.13.1
rotor), resuspended with 100 ml of purifying buffer, centrifuged for another five minutes,
resuspended in another 30ml of purifying buffer and lastly, stored at -80 oC until purification.
Affinity Purification of Mob Bait Proteins
A. Preparation of Appropriate Affinity Columns For Bait Proteins
For GST-tagged proteins, 3ml equivalents of glutathione agarose lyophilized powder (Sigma-
Aldrich, St Louis, MO) were swelled in 42 ml of deionized water at 4oC overnight. The swelled
agarose beads were loaded onto an Econo-Column (Bio-Rad) and washed with ten volumes of 1x
phosphate buffered saline (1x PBS) at pH 7.4. The column was stored with 2mM sodium azide
(company name) in phosphate buffered saline in a cold room. Refer to appendix for 1x PBS
recipes.
For histidine-tagged proteins, 4mls of Ni-NTA agarose (Qiagen, Chatsworth, CA) was
loaded onto an Econo-Column (Bio-Rad) and washed with 10 volumes of wash buffer (same as
lysis buffer) and stored with 2mM sodium azide in a cold room until needed. Refer to the
appendix for wash buffer and lysis buffer recipes.
B. Preparation of Cell Lysate

Lysis buffer containing phenylmethylsulfonyl fluoride (PMSF), benzamidine and
dithiothreitol was added to thawed protein pellets previously stored at -80oC. Pellets were
sonicated and incubated on ice for one minute in an alternating manner until the pellets were
fully resuspended. Additional PMSF was added to the resuspended cells which were
subsequently lysed by the French Press Apparatus (type of press), at 1,200 PSI three times. 2ul
of the crude lysate was placed in 18ul of 5X sample buffer containing -mercaptoethanol
(company name) for further SDS-PAGE gel analysis. PMSF and benzamidine (benzamidine is
only for HsPhocein) was added to the crude lysate, which was then centrifuged at 10,000 rpm
(Beckman JS-13.1 rotor) for thirty minutes at 4oC. 2ul of the supernatant (total soluble fraction)
was added into 18ul of 5x sample buffer with -mercaptoethanol for SDS-PAGE gel analysis.
The recipes for the lysis buffers are listed in Table I in the Appendix. The remaining total
soluble fraction supernatant was diluted to a volume of 120ml with wash buffer containing
appropriate protease inhibitors. The supernatant was then loaded onto appropriate agarose
columns and allowed to pass through the column three times. 6ul of the supernatant flow through
was placed into 14ul of 5x sample buffer with -mercaptoethanol for SDS-PAGE gel analysis.
The column was further washed with wash buffer and stored in 1xPBS containing 2mM sodium
azide. 6ul of the wash flow through and final column slurry were placed into sample buffer for
SDS-PAGE analysis.
In the preparation of the B2Nt cell lysate, cells were thawed from -80 oC, suspended in 150ul
of PMSF and French pressed twice at 1,200 PSI. The cells were then centrifuged at 16000xg for
ten minutes, and the resulting supernatant divided into 15 ml fractions. Each fraction was heat
shocked at 80 oC for twenty minutes (white precipitate was observed). Centrifugation at 16000xg
was conducted for another 10 minutes and the pellet discarded. The lysate was loaded onto a Ni-
NTA column.
Cross-linking GST-tagged Proteins to Glutathione Columns with Dimethyl Pimelimidate
(Aroian Lab Protocols)
To immobilize GST-fusion proteins onto glutathione columns, dimethyl pimelimidate
(DMP, company name), a homobifunctional (containing two imidoester groups) and water-
soluble crosslinking reagent that reacts with primary amines at alkaline pHs was used. First, the
glutathione column resin, saturated with the bait fusion protein was washed with 3 volumes of
200mM HEPES at a pH of 8.5. This was followed by a period of incubation in 5ml of 20mM
DMP in 200mM HEPES for sixty minutes. Residual DMP was subsequently terminated by the
addition of 5mls of 200mM ethanolamine (company name) for thirty minutes. All buffers and
solutions were removed by mild manual centrifugation between new additions of different
crosslinking reagents. 5 ml of 200mM HEPES (company name) was added to the resin and
loaded onto an Econo-Column (BioRad). Finally, the column resin was washed with glycine
elution buffer to remove non-covalently bound molecules. The column was washed with and
stored in 20 ml of 1x TBS (pH 7.5) with 0.05% sodium azide. Free glutathione (company name)
solution runs over the crosslinked column was used to rid of non-crosslinked GST-fusion
proteins. Three different resin samples were placed into 5x sample buffer with -
mercaptoethanol for SDS-PAGE analysis. The three samples included the non-crosslinked resin,
the crosslinked resin, and the crosslinked resin washed with glutathione.
Capturing Potential Mob-interacting Proteins from Xenopus Laevis
4mls of Xenopus Laevis frog extract was thawed, diluted 1:1 with 1x TBS (pH 7.5) and
loaded onto GST-control or 6xHis-control columns four times prior to being passed through
GST-Mob glutathione or 6xHis-tagged-Mob Ni-NTA columns four times. The GST-control and
GST-Mob columns were eluted with 6M guanidinium chloride in 500ul fractions. 6xHis-control
and 6xHis-Mob columns were eluted with 6M magnesium chloride, followed by 4M Urea in
500ul fractions.
Bradford Assay and Dialysis of Eluted Protein Samples
Eluted frog protein fractions from control and fusion protein columns were taken and a
Bradford assay conducted with bovine serum albumin as a protein standard (refer to appendix for
detailed protocol). Fractions with highest concentrations or absorbances were pooled and
dialyzed overnight at 4oC in 1x PBS with 10% glycerol (pH 7.5). Dialyzed samples were
centrifuged for ten minutes in 1ml fractions in the same microfuge tube at maximum speed (type
of centrifuge) until all samples were centrifuged. The supernatant was further concentrated in
Millipore tubes (company name) until the volume was reduced 200ul to 300 ul. 60ul to 75ul of
5x Sample buffer with beta-mercaptoethanol was added to the supernatant, and 250ul of sample
buffer was added to resuspend the flocculent (precipitate after centrifugation). All samples were
then heated at 72oC for five minutes, and stored at -20 oC.
SDS-PAGE
Protein or resin samples in 5x sample buffer with -mercaptoethanol were loaded with
appropriate volumes onto 12.5% polyacrylamide gels and run at 600V, 100W and 20mA using
________ apparatus. A detailed procedure can be found in the appendix.
Results
HsPhocein
Three trials of experiments were conducted to find possible HsPhocein-interacting
proteins from Xenopus laevis.
HsPhocein Interacting Experiments Trial 1
In the crude lysate, total soluble fraction, supernatant flow through, wash flow through
and resin samples of the Mob purification gels (Fig. 1), recurring bands at approximately 54 kDa
and 28kDa can be seen. These bands are especially intense in the crude lysate, total soluble
fraction, and resin samples (Fig. 1). The bands also appear less intense in the supernatant flow
through, and wash flow through samples (Fig.1). Furthermore, other thinner protein bands span
the crude lysate, and total soluble fraction sample lanes (Fig. 1). They however, do not appear in
the supernatant flow through, leaving faint and thin bands at 54kDa and 28kDa (Fig. 1). In the
wash flow through, the 54kDa exists faintly or in some cases disappears (Fig. 1). The resin
sample shows very dark and thick bands at 54kDa and 28kDa (Fig. 1A).
In the GST-control and GST-fusion crosslinking gels, bands at 54kDa and 28 kDa are
apparent in both the DMP crosslinked and non-crosslinked resins (Fig.2). The 54kDa and 28kDa
bands in the non-crosslinked resin are thicker than those of the crosslinked resin (Fig.2). In the
HsMob2 and trial 3 HsPhocein crosslinking samples, the 28kDa and 54kDa bands in the
glutathione washed resin are thinner than the crosslinked resins (Fig.2 ). The same bands in
the glutathione washed resin in trial 2 HsPhocein experiments appear thicker than the crosslinked
resin (Fig. 2 ). For the GST-controls in trial 3 HsPhocein experiments, the GST band appears
around 54kDa to 56kDa, in the crosslinked resin since GST appear as dimers rather than
monomers.
In the trial one HsPhocein interacting SDS-PAGE gel, 54kDa and 28kDa bands are
visible in the storage buffer, crosslinked resin, resin following loading of the Xenopus laevis frog
extract, and the resin following 6M GnCl elution (Fig.1C). The 54kDa and 28kDa band in the
resin, after elution with GnCl are slightly thinner than those of the resin prior to elution. In the
flocculent and supernatant samples, bands different from those of 54kDa and 28kDa bands are
noticeable, and are indicated by the red boxed bands on the gel image (Fig.1C).
Figure 1: SDS-PAGE gels of samples from Trial 1 GST-HsPhocein Interacting Experiments. GST-HsPhocein purification,
crosslinking and pull down assay experiment results represented in 12.5% polyacrylamide gels. The bait protein was HsPhocein
and the prey proteins were possible Mob-interacting proteins that interacted with HsPhocein. The crosslinking and purification
gels were stained in coomassie blue (A and B) while the interacting gel was stained in colloidal coomassie (C).
_________________________________________________________________________________________________________________________________________________
A)HsPhocein purification SDS-PAGE gel

1 Prestain markers
2 Low molecular weight makers
3 GST-XM-03 crude extract
5 GST-XM-03 total soluble fraction (supernatant)
6 GST-XM-03 diluted total soluble fraction flow through
7 Washing buffer flow through
8 Column resin sample buffer supernatant
Gel Run Time: 1 hour and 15 minutes

600V, 100W, 20mA
1 2 3 4 5 6 7
B) GST-Phocein (XM-03) DMP crosslinking SDS-PAGE
gel
1 Prestain markers
54kDa bands 2 Low molecular weight makers
3 Column GST-XM-03 non-cross-linked resin
4 Column GST-XM-03 non-cross-linked resin
5 Sample Buffer
28kDa bands
6 Column GST-XM03-DMP cross-linked resin
7 Column GST-XM03-DMP cross-linked resin

600V, 100W, 20mA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 C) HsPhocein-interacting protein SDS-PAGE gel

97 1 Low Molecular Weight Markers
2 Column Storage Buffer
60 3 Column Storage Buffer
4 Crosslinked GST-XM-03column resin
45 5 Resin after loading of frog extract
6 PBS column wash flow through
30 7 Resin after PBS wash
8 Resin after elution with 6M GnCl
9 PBS column wash after elution with GnCl
10 Low molecular weight markers
11 Flocculent of GnCl eluted samples
12 Supernatant of GnCl eluted samples
13 Filtered waste

HsPhocein Interacting Experiment Trial 2
In the trial two HsPhocein purification gel, the crude lysate, total soluble fraction, and
column resin samples show relatively thick and intense bands at 54kDa and 28kDa (Fig.2A). The
supernatant flow through shows fainter bands at 54kDa and 28kDa, while the wash flow through
shows even fainter bands at those molecular weights (Fig.2A). Other thinner and less intense
bands also span each sample lane with the exception of the wash flow through (Fig.2A). In the
supernatant flow through, however, these bands are smaller and less intense (Fig. 2A). Some of
these bands also reappear in the resin sample (Fig. 2B). The trial two GST-control purification gel
displays similar gel bands at 54kDa and 28kDa in the crude, total soluble fraction, and resin
samples (Fig. 2B). The supernatant flow show smaller bands at these sizes and the wash flow
through show no signs of the same bands (Fig.2B).
The trial 2 crosslinking gel of samples taken from GST-HsPhocein and GST-control
columns show the difference in resin samples during three important steps in the crosslinking
procedure. Three resin samples were taken from each column: 1) resin before crosslinking, 2)
resin after crosslinking and 3) resin after crosslinking and washed with free glutathione. In the
lanes corresponding to the GST-control column, a thick and intense protein band at approximately
28kDa can be seen. Once crosslinked, the 28kDa band decreases in size (Fig. 2C). The band then
increases in size once it was washed with glutathione, to be even thicker than the non-crosslinked
resin band (Fig. 2C). The exact same trend can be observed with the intense bands at 56kDa and
28kDa in the GST-HsPhocein resin samples (Fig. 2C).
Interacting gel one of trial two HsPhocein experiments compared the difference between
GST-HsPhocein eluted samples with those of the GST-control (Fig. 2D). Protein bands that exist
in the GST-HsPhocein flocculent and supernatant that are different from protein bands that exist in
the GST-control flocculent and supernatant can be identified by a red box on the image (Fig. 2D).
The GST-control lanes (2-5, Fig, 2D) also consist of very discrete and separated bands while the
GST-HsPhocein lanes show low resolution and smearing of protein bands, even when lower
concentrations of protein were loaded (7-12, Fig. 2D). Furthermore, the GST-HsPhocein column
revealed large and intense 28kDa and 54kDa bands (Fig. 2D).
The trial two interacting gel two is a gel that compares the GST-HsPhocein eluted
samples with the GST-HsPhocein glutathione washed crosslinked resin (Fig. 2E). A majority of
the bands that are present in the flocculent and supernatant are identical to those that appear in the
glutathione washed crosslinked resin (blue boxed, Fig. 2E).
Figure 2: SDS-PAGE gels of samples from Trial 2 GST-HsPhocein Interacting Experiments. GST-control and GST-XM03
purification, crosslinking and pull down assay 12.5% polyacrylamide gels are displayed. The bait is HsPhocein and the
prey are possible HsPhocein-interacting proteins. The crosslinking and purification gels were stained in coomassie blue (A,
B and C) while the interacting gels were stained in colloidal coomassie stain (D, and E)
_________________________________________________________________________________________________________________________________________
200 1 2 Interacting
HsPhocein 3 4 Experiment
5 6 7Trial
8 2
A) HsPhocein purification SDS-PAGE gel
3 GST-XM-03 total soluble fraction
50 ____________________________54kDa 4 GST-XM-03 supernatant flow through
40 5 Washing buffer flow through
__
6 PBS Wash flow through
30 7 Column resin
28kDa 8 Low molecular weight markers
25
20
15 Gel Run Time: 1 hour and 30 minutes
10
200 1 2 3 4 5 6 7 8 9 10
11 12 13 14 B) GST-control purification SDS-PAGE gel
2 GST-control crude extract
3 GST-control total soluble fraction
60 4 Empty
50 5 GST-control supernatant flow through
40 56 kDa 6 Washing buffer flow through
7 PBS Wash flow through
30 8 Empty
9 Empty
25 28kDa 10 Column resin
20
15
10
Figure 2: continued
_________________________________________________________________________________________________________________________________________
1 2 3 4 5 6 7 8 9 10 C) GST-control and HsPhocein Crosslinking SDS-PAGE

200
gel
2 GST-control resin before crosslinking with DMP
54kDa bands 3 GST-control resin after crosslinking with DMP
50
4 GST-control resin after glutathione wash
40
5 Empty
6 Empty
30 ________ 7 Low molecular weight makers
28kDa bands
8 GST-HsPhocein resin before crosslinking with DMP
25 9 GST-HsPhocein resin after crosslinking with DMP
20 28kDa bands
10 Column resin
10
1 2 3 4 5 6 7 8 9 10 11 12
D) Hs Phocein-interacting SDS-PAGE gel 1 of GnCl
200 13 14 eluted protein samples from GST-control and GST-
HsPhocein columns
2 GST-control-flocculent of GnCl eluted samples(5ul)
50
54kDa bands 3 GST-control- flocculent of GnCl eluted samples (10ul)
40 4 GST-control- supernatant GnCl eluted samples(5ul)
5 GST-control supernatant of GnCl eluted samples (10ul
30 6 Low molecular weight markers
28kDa bands 7 GST-HsPhocein-flocculent of GnCl eluted samples(3ul)
25 8 GST-HsPhocein- flocculent of GnCl eluted samples (6ul)
10 GST-HsPhocein supernatant GnCl eluted samples(5ul)
15 11GST-HsPhocein supernatant GnCl eluted samples(7ul)
12 GST-HsPhocein supernatant GnCl eluted samples(10ul)
10
Gel Run Time: 1hour and 15 minutes
200
1 2 3 4 5 6 7 8 9 10 11 12 E) HsPhocein-interacting SDS-PAGE gel 2 of resin after
glutathione wash and GnCl eluted protein samples
from GST-HsPhocein
1 Low molecular weight marker
) GST-HsPhocein-flocculent of GnCl eluted samples(10ul)
2
50 Gel
3 Run Time: 1 hour
GST-HsPhocein- and 15 of
flocculent minutes
GnCl eluted samples (7ul)
40 600V,
4 100W, 20mAflocculent of GnCl eluted samples (5ul)
GST-HsPhocein-
5 GST-HsPhocein- flocculent of GnCl eluted samples (2ul)
30 6 GST-HsPhocein-resin after glutathione wash (5ul)
7 GST-HsPhocein-resin after glutathione wash (10ul)
20 9GST-HsPhocein supernatant GnCl eluted samples(5ul)
10GST-HsPhocein supernatant GnCl eluted samples(7ul)
15 11 GST-HsPhocein supernatant GnCl eluted samples(10ul)
10
Gel Run Time: 1hour and 5 minutes
HsPhocein Interacting Experiments Trial 3
In the trial three GST-control purification gel, the crude lysate and total soluble fraction
reveal a 28kDa which becomes smaller in the supernatant flow through and extremely faint in
the wash flow through (Fig. 3A). Other protein bands also span sample lanes except in the wash
flow through and resin (Fig. 3A). Unlike the GST-control purification in trial 2, bands at 56kD
are thinner (Fig. 3A). In the GST-HsPhocein lanes, dark bands at 54kDa occur in the crude
lysate, total soluble fraction, and column resin (Fig. 3A). The band becomes less intense in the
supernatant flow through and almost disappears in the wash (Fig. 3A). The 28kDa band is less
prominent here, than in trial one and two GST-HsPhocein purifications (Fig. 1A, 2A, B and 3A).
The GST-control and GST-HsPhocein crosslinking gel in trial 3 produced different gel
patterns than those in trial 2 (Fig. 2C). With the GST-control, the non-crosslinked resin shows
an intense band at 28kDa (Fig. 3B). Once crosslinked, the 28kDa band almost disappears while a
54kDa band appears (Fig. 3B). In the crosslinked resin washed by glutathione, the 28kDa band
reappears, and the 54kDa band becomes faint (Fig. 3B). Although the 28kDa band reappears in
the crosslinked resin washed by glutathione, the band is much thinner than the band in the non-
crosslinked resin (Fig. 3B). In the GST-HsPhocein crosslinking, a band at 54kDa appears in the
non-crosslinked resin and decreases in thickness and intensity following crosslinking, A 28kDa
also appears in the resin after crosslinking (Fig. 3B). After washing with glutathione, there is
reappearance of the 54kDa band, and an extremely faint 28kDa band (Fig.3B). The band at
54kDa is also thinner than the same band in the non-crosslinked resin (Fig.3B). Recall that in
trial 2 the glutathione washed resins have 54kDa or 28kDa bands that are thicker than the non-
crosslinked resins in both the GST-HsPhocein and the GST-control samples (Fig. 2C).
In the trial 3 interacting gel, discrete bands in the GST-HsPhocein supernatant are
different from the GST-control flocculent and supernatants, as well as the GST-control and GST-
HsPhocein glutathione washed crosslinked resins (red boxed bands, Fig. 3C).
The boxed protein bands from the HsPhocein supernatant in figure 3C were chosen as
good candidates for possible HsPhocein interacting frog proteins. Mass spectrometry results
identified the peptide sequences for each band. Each peptide was contained in the full sequences
of protein phosphatise 2A, phocein and calpain 3. Mascot mowse probability distributions
indicate that a score above 32 is a search of significance (Fig. 3D). A multiple alignment
sequence was conducted with HsPhocein and the frog phocein sequence and yielded a score of
97 similar (Fig. 3E).

Figure 3: SDS-PAGE gels of samples from Trial 3 GST-HsPhocein Interacting Experiments. GST-control and GST-XM03
purification, crosslinking and pull down assay 12.5% polyacrylamide gels are displayed. The bait is HsPhocein and the prey are
possible HsPhocein-interacting proteins. The crosslinking and purification gels were stained in coomassie blue (A, and B) while
the interacting gel was stained in colloidal coomassie stain (C). Samples from different columns are described in the captions by :
Column from which samples came from-type of sample. Panel D and E show mass spectrometry results and sequence alignments.
________________________________________________________________________________________________________________________________________________
1 2 3 4 5 6 7 8 9 10 11 12 A) GST-HsPhocein and GST-control purification SDS-

200 PAGE gel
2 GST-control- crude extract
3 GST-control-total soluble fraction
50 54kDa bands 4 GST-control-supernatant flow through
40 5 GST-control wash flow through
6 GST-control-column resin
30 7 Empty
28 kDa bands 28 kDa bands 8 GST-HsPhocein- crude extract
9 GST-HsPhocein- total soluble fraction
25
10 GST-HsPhocein- supernatant flow through
20
11 GST-HsPhocein-Washing buffer flow through
12 GST-HsPhocein- column resin
15
10
200 1 2 3 4 5 6 7 8
B) GST-control and GST-HsPhocein Crosslinking SDS-
9 10 11 12 PAGE gel
2 GST-control-column resin before crosslinking
50 3 GST-control-column resin after crosslinking before
40
54kDa bands
glutathione wash
4 GST-control-column resin after glutathione wash
30 5 Empty
6 GST-HsPhocein-column resin before crosslinking
25 28kDa bands 7 GST-HsPhocein-column resin after crosslinking before
20 glutathione wash
8 GST-HsPhocein-column resin after glutathione wash
15
10
200 1 2 3 4 5 6 7 8 9 10 11 12 C) GST-HsPhocein interacting SDS-PAGE gel of GnCl

eluted samples GST-control and GST-Phocein columns
2 GST-control-flocculent of GnCl eluted sample (10ul)3
GST- control-flocculent of GnCl eluted samples(5ul)
4 GST-control-column resin after crosslinking and
50
glutathione wash
40
5 GST-control-supernatant of GnCl eluted sample (5ul)
6 GST-control-supernatant of GnCl eluted sample (10ul)
30 7 Low molecular weight markers
8 GST-HsPhocein- flocculent of GnCl eluted sample (10ul)
25 9 GST-HsPhocein- flocculent of GnCl eluted sample (5ul)
20 10 GST-HsPhocein-column resin after crosslinking and
glutathione wash
15 11 GST-HsPhocein- supernatant of GnCl eluted sample (5ul)
12 GST-HsPhocein- supernatant of GnCl eluted sample
10 (10ul)
Gel Run time: 1 hour and 40 minutes
Figure 3: Continuation
_________________________________________________________________________________________________________________________________________
D) Two important Mascot searches of interest from mass spectrometry analysis
MASCOT SEARCHES
SEARCH 1
gi|148222419 Mass: 81430 Score: 37 Queries matched: 1 emPAI: 0.05
calpain 3, (p94) [Xenopus laevis]
Peptide Sequence: K.DLQSGIHIK.K
Full Sequence of Search 1
>gi|148222419|ref|NP_001087053.1| calpain 3, (p94) [Xenopus laevis]
.....VQQFSRIIICSPTLDFLNWGSDQTSWHKTAYKNIWPKECGLEDSFNKDLFCKNPQYLIWITPSDEVKKGY
NVIISLMQNHRNRQKFGHEWLPIGFLVSKVGAELPDPQMKMPGSFFPKDLQSGIHIKKRRDVTKGFRLAA
GRYVITPFNADRNQESSFLLQVFLRSQGCTVELGDSQSFLRNEEKTHEDIFMKYATQGGKMNARDLQKFL....
SEARCH 2
hypothetical protein LOC496323 [Xenopus laevis]
Peptide sequence: R.IFSHAYFHHR.Q (sequenced from gel bands shown in panel C)
Full sequence of hypothetical protein given by search 2

1 mvmaegtavl rrnrpgtktq dfynwpdesf eemdstlavq qyiqqnirtd csnidkildp
61 pegqdegvwk yehlrqfcle lnglavklqt echpdtctqm tateqwiflc aahktpkecp
121 aidytrhtld gaacllnsnk yfpsrvsike ssvaklgsvc rriyrifsha yfhhrqifde
181 yenetflchr ftkfvmkynl mskdnlivpi leeevqnsva gesea
SEARCH 3
protein phosphatase 2, catalytic subunit, alpha isoform [Xenopus tropicalis]
Peptide Sequence: K.YSFLQFDPAPR.R
>gi|53749698|ref|NP_001005443.1| protein phosphatase 2, catalytic subunit, alpha isoform
[Xenopus tropicalis]
MEDKTFTKELDQWIEQLNDCKQLNESQVRTLCEKAKEILTKESNVQDVRCPVTVCGDVHGQFHDLMELFR
IGGKSPDTNYLFMGDYVDRGYYSVETVTLLVALKVRYPERITILRGNHESRQITQVYGFYDECLRKYGNA
NVWKYFTDLFDYLPLTALVDGQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWG
ISPRGAGYTFGQDISETFNHANGLTLVSRAHQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDD
TLKYSFLQFDPAPRRGEPHVTRRTPDYFL
Highlighted region shows the peptide sequence given from mass spectrometry analysis.
*The Mascot score distribution indicated that a significant search would have a score of
greater than 32.
________________________________________________________________________________________________________________________________________________
E) Multiple sequence alignment of human phocein, phocein in Xenopus laevis and the full sequence given in mascot
search. The sequence from the mascot search is identical to the Xenopus laevis sequence (same protein)
Sequence Source
gi|41349449|ref|NP_056202.2| Homo sapiens
gi|148228740|ref|NP_001088946 Xenopus laevis
SeqA Name Len(aa) SeqB Name Len(aa) Score

===================================================================================================
1 gi|148228740|ref|NP_001088946. 225 2 gi|41349449|ref|NP_056202.2| 225 97
===================================================================================================
gi|148228740|ref|NP_001088946. MVMAEGTAVLRRNRPGTKTQDFYNWPDESFEEMDSTLAVQQYIQQNIRTD 50
gi|41349449|ref|NP_056202.2| MVMAEGTAVLRRNRPGTKAQDFYNWPDESFDEMDSTLAVQQYIQQNIRAD 50
******************:***********:*****************:*
gi|148228740|ref|NP_001088946. CSNIDKILDPPEGQDEGVWKYEHLRQFCLELNGLAVKLQTECHPDTCTQM 100

gi|41349449|ref|NP_056202.2| CSNIDKILEPPEGQDEGVWKYEHLRQFCLELNGLAVKLQSECHPDTCTQM 100
********:******************************:**********
gi|148228740|ref|NP_001088946. TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVSIKE 150

gi|41349449|ref|NP_056202.2| TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVSIKE 150
**************************************************
gi|148228740|ref|NP_001088946. SSVAKLGSVCRRIYRIFSHAYFHHRQIFDEYENETFLCHRFTKFVMKYNL 200

gi|41349449|ref|NP_056202.2| SSVAKLGSVCRRIYRIFSHAYFHHRQIFDEYENETFLCHRFTKFVMKYNL 200
**************************************************
gi|148228740|ref|NP_001088946. MSKDNLIVPILEEEVQNSVAGESEA 225

gi|41349449|ref|NP_056202.2| MSKDNLIVPILEEEVQNSVSGESEA 225
*******************:*****
Highlighted region is the peptide sequence provided by mass spectrometry analysis of gel bands cut
out from the gel shown in panel C
HsMob2 Interacting Experiment
The GST-control purification gel in the HsMob2 interacting experiment shows intense
bands at 28kDa and 54kDa in the crude extract, and total insoluble fractions (Fig. 4A). Only the
54kDa band is apparent in the supernatant flow through, and no bands are seen in the wash (Fig.
4A). The non-crosslinked resin shows a dark band at 28kDa, which becomes thinner after being
crosslinked and washed with glutathione (Fig. 4A).
In the GST-HsMob2 purification gel, intense and thick bands at 54kDa and 28kDa are
seen in the crude lysate, total soluble fraction, supernatant flow through and column resin (Fig.
4B). The crude lysate bands at these sizes are the thickest, followed by the total soluble fraction
and then the supernatant flow through (Fig. 4B). The wash flow through shows no evidence of
protein bands (Fig. 4B). Furthermore, the non-crosslinked resin show thicker bands at 54kDa and
28kDa than the glutathione washed crosslinked resin (Fig. 4B).
The GST-HsMob2 interacting gel 2 show red boxed protein bands in the flocculent eluted
from the GST-HsMob2 column (Fig 4D). These bands are not present in the GST-control
flocculent or supernatants, nor the glutathione washed crosslinked resins (Fig. 4Cand D). These
bands were chosen as good candidates of possible HsMob2 interacting frog proteins. The peptide
sequences of these bands were determined by mass spectrometry and were found to be contained
in the full sequences of liprin beta-1, bone morphogenetic protein 7 and Mob2 (Fig. 4E).
Sequence alignments of phocein from human and frogs, as well as the full sequence of
unknown protein were conducted and resulted in scores of 96 when aligning the HsPhocein with
Xenopus tropicalis, 97 when aligning HsPhocein with, and 98 when aligning the two frog phocein
sequences and 13 when aligning the unknown protein with all the other phocein sequences (Fig.
4F).
Figure 4: SDS-PAGE gels of samples GST-HsMob2 Interacting Experiments. GST-control and GST-HsMob2 purification,
crosslinking and pull down assay 12.5% polyacrylamide gels are displayed. The bait is GST-HsMob2 and the prey are possible
HsMob2-interacting proteins. The crosslinking and purification gels were stained in coomassie blue (A, and B) while the
interacting gels were stained in colloidal coomassie stain (D). Samples from different columns are described in the captions by :
Column from which samples came from-type of sample. Panel E and F show mascot searches for possible HsMob2 interacting
proteins and sequence alignments for frog and human phoceins with the unknown protein sequence.
_______________________________________________________________________________________________________________________________________________
200 1 2 3 4 5 6 7 8 A) GST-control SDS-PAGE purification gel

2 GST-control- crude extract
3 GST-control-total soluble fraction
50 4 GST-control-supernatant flow through
5 GST-control wash flow through
40 6 Empty
7 GST-control-column resin crosslinked with DMP after
30 glutathione wash
8 GST-control-column resin before crosslinking with DMP
28kDa bands
25
20
15
10
B) GST-HsMob2 SDS-PAGE purification gel

2GST-HsMob2- crude extract (5ul)
3 GST-HsMob2- crude extract (10ul)
4 GST-HsMob2- total soluble fraction (5ul)
5 GST-HsMob2- total soluble fraction (10ul)
6 GST-HsMob2- supernatant flow through (5ul)
7 GST-HsMob2- supernatant flow through (10ul)
8 GST-HsMob2- wash flow through (5ul)
9 GST-HsMob2- wash flow through (10ul)
10 GST-HsMob2- column resin before crosslinking
11 GST-HsMob2- column resin after crosslinking
1 2 3 4 5 6 200 7 8 9 10 11 12 C) GST-HsMob2 interacting SDS-PAGE gel 1 of GnCl

eluted samples GST-control and GST-Phocein columns
2 GST-HsMob2-flocculent from GnCl eluted samples (5ul)
50 4 GST-HsMob2-column resin after crosslinking and
glutathione wash
40 5 GST-HsMob2-supernatant from GnCl eluted samples (5ul)
6 GST-HsMob2-supernatant from GnCl eluted samples (10ul)
30 7 Low molecular weight marker
8 GST-control-flocculent from GnCl eluted samples (5ul)
25 9 GST-control-flocculent from GnCl eluted samples 105ul)
20 10 GST-control-column resin after crosslinking and
glutathione wash
15 11 GST-control- supernatant from GnCl eluted samples (5ul)
12 GST-control- supernatant from GnCl eluted samples
10 (10ul)

Figure 4: SDS-PAGE gels of samples GST-HsMob2 Interacting Experiments. GST-control and GST-HsMob2 purification,
crosslinking and pull down assay 12.5% polyacrylamide gels are displayed. The bait is GST-HsMob2 and the prey are possible
HsMob2-interacting proteins. The crosslinking and purification gels were stained in coomassie blue (A, and B) while the
interacting gels were stained in colloidal coomassie stain (D). Samples from different columns are described in the captions by :
Column from which samples came from-type of sample. Panel E shows mascot searches for possible HsMob2 interacting proteins.
_______________________________________________________________________________________________________________________________________________
D) GST-HsMob2 interacting SDS-PAGE gel 2 of GnCl eluted

1 2 3 4 5 6 200 7 8 9 10 11 samples GST-control and GST-Phocein columns
1Low molecular weight marker
2 GST-control-flocculent from GnCl eluted samples (5ul)
3 GST-control-flocculent from GnCl eluted samples 105ul)
4 GST-control- supernatant from GnCl eluted samples (5ul)
5 GST-control- supernatant from GnCl eluted samples (10ul)
50 6 Empty
40 8 GST-HsMob2-flocculent from GnCl eluted samples (5ul)
30 10 GST-HsMob2-column resin after crosslinking and glutathione
wash
11 GST-HsMob2-supernatant from GnCl eluted samples (5ul)
25 12GST-HsMob2-supernatant from GnCl eluted samples (10ul)
20
15
10
E) Three important Mascot searches of interest from mass spectrometry analysis
SEARCH 1
Unknown (protein for MGC:81135) [Xenopus laevis] Peptide Sequence: K.LVTDEDVFPTK.Y
Full sequence of search 3:
msirrsgsyt vqkkskgkpn gkkpaseekk lylepeytrv rvtdvefkql vtlpqeidln
ewlasnittf fnhinlqyst isefctgetc qtmaacntqy ywydergkkv kctapqyidf
vmssiqklvt dedvfptkyg refpssfesl vkkicrylfh vvahiywahf keitvlelhg
hlntlfihfl lfvrefslld pketsvlddl seilfseenr eavgatgeaq nhvker
SEARCH 2
gi|147900035 Score: 41 Queries matched: 2
bone morphogenetic protein 7 [Xenopus laevis]Peptide Sequence: -.MNVLQKNK.T
>gi|147900035|ref|NP_001080866.1| bone morphogenetic protein 7[Xenopus laevi]s]
MNVLQKNKTGSVLLWTYFLWRIILADFTLDNEVHSSFIQRRLRGQERREMQREILSILGLPHRPRPHLYG
KQNSAPMFMLDLYNAMTVEEEEAEGFSYPYKPIFTTQGPPLATQQDSNFLNDADMVMSFVNLVEHDKEFF
HQRRHQREFRFDLAKIPEGEAVTAAEFRIYKDYIRERFENETFQISVYQVLQEHQGRDSDLYELDSRTIW.......
SEARCH 3
similar to PTPRF interacting protein, binding protein 1 (liprin beta 1)
[Xenopus laevis]
Peptide Sequence: K.DTEGLVQEMNELR.L
Full Sequence of Search 4:
>gi|147903473|ref|NP_001082516.1| similar to PTPRF interacting protein, binding protein
1 (liprin beta 1) [Xenopus laevis]
.........RGQLPDSTADVLVEWLQSHMVNGHISGSTDICQERLARLENDKESFVLQVSVLTDQVEAQGEKIRDLEFC
LEEHREKLNDTEEMLQQELLSRTSLESQKLDLMAEISNLKLKLVSVEKDRKEFEERYKDTEGLVQEMNELRLRVL.............
Highlight shows the peptide sequence given from mass spectrometry results
*The Mascot score distribution indicated that a significant search would have a score of greater than 32.
Figure 4: Continued
_______________________________________________________________________________________________________________________________________________
SEARCH 5
RAP1A, member of RAS oncogene family [Xenopus laevis]
Peptide Sequence: K.INVNEIFYDLVR.Q
>gi|147900077|ref|NP_001084500.1| RAP1A, member of RAS oncogene family [Xenopus laevis]
MREYKLVVLGSGGVGKSALTVQFVQGIFVEKYDPTIEDSYRKQVEVEGQQCMLEILDTAGTEQFTAMRDL
YMKNGQGFALVYSITAQSTFNDLQDLREQILRVKDTEDVPMILVGNKCDLEDERVVGKEQGHNLARQWNN
CAFLESSAKSKINVNEIFYDLVRQINRKTPVEKKKPSKKPKCLLL
F) A sequence alignment of human and frog Phocein protein FASTA sequences using CLUSTAL W multiple sequence alignment.
Parameters were set at default.
Sequence Source
gi|41349449|ref|NP_056202.2| Homo sapiens
gi|62857563|ref|NP_001017210.1 Xenopus tropicalis
gi|148228740|ref|NP_001088946 Xenopus laevis
gi|47122919|gb|AAH70585.1| Unknown protein from mascot search containing peptide sequence.
SeqA Name Len(aa) SeqB Name Len(aa) Score

============================================================================================================
1 gi|41349449|ref|NP_056202.2| 225 2 gi|62857563|ref|NP_001017210.1 225 96
1 gi|41349449|ref|NP_056202.2| 225 3 gi|148228740|ref|NP_001088946. 225 97
1 gi|41349449|ref|NP_056202.2| 225 4 gi|47122919|gb|AAH70585.1| 236 13
2 gi|62857563|ref|NP_001017210.1 225 3 gi|148228740|ref|NP_001088946. 225 98
2 gi|62857563|ref|NP_001017210.1 225 4 gi|47122919|gb|AAH70585.1| 236 13
3 gi|148228740|ref|NP_001088946. 225 4 gi|47122919|gb|AAH70585.1| 236 13
gi|62857563|ref|NP_001017210.1 MVMAEGTVVLRRNRPGTKAQDFYNWPDESFEEMDSTLAVQQYIQQNIRTD 50
gi|148228740|ref|NP_001088946. MVMAEGTAVLRRNRPGTKTQDFYNWPDESFEEMDSTLAVQQYIQQNIRTD 50
gi|41349449|ref|NP_056202.2| MVMAEGTAVLRRNRPGTKAQDFYNWPDESFDEMDSTLAVQQYIQQNIRAD 50
gi|47122919|gb|AAH70585.1| -------MSIRRSGSYTVQKKSKGKPNGKKPASEEKKLYLEPEYTRVRVT 43
:**. . * :. . *: . :.. : .:*.
gi|62857563|ref|NP_001017210.1 CSNIDRILDPPEGQDEGVWKYEHLRQFCLELNGLAVKLQTECHPDTCTQM 100

gi|148228740|ref|NP_001088946. CSNIDKILDPPEGQDEGVWKYEHLRQFCLELNGLAVKLQTECHPDTCTQM 100
gi|41349449|ref|NP_056202.2| CSNIDKILEPPEGQDEGVWKYEHLRQFCLELNGLAVKLQSECHPDTCTQM 100
gi|47122919|gb|AAH70585.1| DVEFKQLVTLPQEIDLNEWLASNITTFFNHINLQYSTISEFCTGETCQTM 93
::.::: *: * . * .:: * .:* .:. * :** *
gi|62857563|ref|NP_001017210.1 TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVS--I 148

gi|148228740|ref|NP_001088946. TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVS--I 148
gi|41349449|ref|NP_056202.2| TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVS--I 148
gi|47122919|gb|AAH70585.1| AACNTQYYWYDERGKKVKCTAPQYIDFVMSSIQKLVTDEDVFPTKYGREF 143
:* : : : . :*.* :* ..:.. *:..:. **:: . :
gi|62857563|ref|NP_001017210.1 KESSVAKLGSVCRRIYRIFSHAYFHHR---QIFDEYENETFLCHRFTKFV 195

gi|148228740|ref|NP_001088946. KESSVAKLGSVCRRIYRIFSHAYFHHR---QIFDEYENETFLCHRFTKFV 195
gi|41349449|ref|NP_056202.2| KESSVAKLGSVCRRIYRIFSHAYFHHR---QIFDEYENETFLCHRFTKFV 195
gi|47122919|gb|AAH70585.1| PSSFESLVKKICRYLFHVVAHIYWAHFKEITVLELHGHLNTLFIHFLLFV 193
.* : : .:** ::::.:* *: * ::: : : . * :* **
gi|62857563|ref|NP_001017210.1 MKYNLMSK------DNLIVPILEEEVQNSVAGESEA------- 225

gi|148228740|ref|NP_001088946. MKYNLMSK------DNLIVPILEEEVQNSVAGESEA------- 225
gi|41349449|ref|NP_056202.2| MKYNLMSK------DNLIVPILEEEVQNSVSGESEA------- 225
gi|47122919|gb|AAH70585.1| REFSLLDPKETSVLDDLSEILFSEENREAVGATGEAQNHVKER 236
::.*:. *:* ::.** :::*.. .**
Peptide sequence obtained from the protein gel bands of possible HsMob2 interacting frog proteins is highlighted
HsMobLak Interacting Experiment
The crude lysate, total soluble fraction, and resin sample in the 6xHis-B2Nt purification
samples show intense bands at 13kDa. The supernatant flow through, and wash flow through do
not contain the 13kDa band (Fig. 5A). The purification of 6xHis-HsMobLak also show similar
patterns where the crude lysate, total soluble fraction and resin contain intense bands at 26kDa
and very faintly in the supernatant and wash flow through (Fig. 5A). In the 6xHis-HsMobLak
interacting gel, the MgCl2 samples from the HsMobLak column show protein bands that do not
appear in eluted samples from the 6xHis-B2Nt control column (red boxes, Fig. 5B). These bands
were chosen as good candidates for HsMobLak interacting frog proteins and were sent for mass
spectrometry analysis.
The bands that were sent for MS analysis yielded three significant peptide sequences that
were each contained in the full sequences of retinoblastoma binding protein 7, nucleoplasmin,
and Y-box-binding protein 2-A. These proteins all exist in Xenopus laevis. A multiple sequence
alignment of Xenopus laevis MobLak and HsMobLak was also conducted and resulted in an identity score
of 83.
Figure 5: SDS-PAGE gels of samples from 6xHis-HsMobLak Interacting Experiments. 6xHis-HsMobLak and 6xHis-B2Nt purification, and pull
down assay 12.5% polyacrylamide gels are displayed. The bait is 6xHis-HsMobLak and the prey are possible HsMoblak-interacting proteins.
The control is 6xHis-B2Nt. Samples from different columns are described in the captions by : Column from which samples came from-type of
sample. C) Mass spectrometry peptide sequences and mascot results. D) A sequence alignment of HsMobLak and Xenopus laevis MobLak.
______________________________________________________________________________________________________________________________________________
1 2 3 4 5 6 7 8 9 10 11 12
A) 6xHis-HsMobLak and 6xHis-B2Nt SDS-PAGE
purification gel
1 Prestain Marker
2 6xHis-B2Nt-crude extract
3 6xHis-B2Nt-total soluble fraction
4 6xHis-B2Nt-supernatant flow through
5 6xHis-B2Nt-wash flow through
6 6xHis-B2Nt-column resin sample
7 Empty
8 6xHis-HsMobLak-crude extract
9 6xHis-HsMobLak-total soluble fraction
10 6xHis-HsMobLak-supernatant flow through
26 kDa bands 11 6xHis-HsMobLak-wash flow through
13kDa bands 12 6xHis-HsMobLak-column resin sample
B) 6xHis-HsMobLak interacting SDS-PAGE gel of 6M

1 2 3 4 5 6 7 8 10 11
MgCl2 and 4MUrea eluted samples deom
170 6xHis-HsMobLak and 6xHis-B2Nt columns
1 Prestain marker
2 6xHis-B2Nt- MgCl2 eluted sample (5ul)
3 6xHis-B2Nt- MgCl2 eluted sample (10ul)
55 4 6xHis-B2Nt- Urea eluted sample (5ul)
5 6xHis-B2Nt- Urea eluted sample (10ul)
40 6 Empty
7 Empty
35 8 6xHis-HsMobLak- MgCl2 eluted sample (5ul)
9 6xHis-HsMobLak- MgCl2 eluted sample (10ul)
25 10 6xHis-HsMobLak- Urea eluted sample (5ul)
11 6xHis-HsMobLak- Urea eluted sample (10ul

_______________________________________________________________________________________________________________________________________________________________
E) Three important Mascot searches of interest from mass spectrometry analysis
SEARCH 1
retinoblastoma binding protein 7 [Xenopus laevis]
Mass Spectrometry Peptide Sequence of highest score: K.TVALWDLR.N

>gi|160420243|ref|NP_001080364.1| retinoblastoma binding protein 7 [Xenopus laevis]
MANKEMFEDTVEERVINEEYKIWKKNTPFLYDLVMTHALEWPSLTVQWLPDVTRPEGKDYALHWLVLGTH
TSDEQNHLVVARVQVPNDDAQFDASHYDSEKGEFGGFGSVSGKIETEIKINHEGEVNRARYMPQNPCIIA
TKTPSADVLVFDYTKHPSKPDPSGECSPDLRLRGHQKEGYGLSWNSNLSGHLLSASDDHTVCLWDISAGP
KEGKVVDAKAVFTGHSAVVEDVAWHLLHESLFGSVADDQKLMIWDTRSNTTSKPSHSVDAHTAEVNCLSF
NPYSEFILATGSADKTVALWDLRNLKLKLHSFESHKDEIFQVHWSPHNETILASSGTDRRLNVWDLSKIG
EEQSAEDAEDGPPELLFIHGGHTAKISDFSWNPNEPWVICSVSEDNIMQIWQMAENIYNDEEPDIPASEL
EAQGS
SEARCH 2
nucleoplasmin [Xenopus laevis]
Mass Spectrometry Peptide Sequence of highest score: K.AKDEFHIVEIVTQEEGK.E

>gi|833629|emb|CAA68363.1| nucleoplasmin [Xenopus laevis]
NTSKVEKPVSLIWGCELNEQNKTFAFKIEDEEEKCEHQLALRTVCLGDKAKDEFHIVEIVTQEEGKEKPV
PIASLKPSILPMATMVGIELTPPVTFRLKAGSGPVYISGQHVAMEEDYSWAEEEDEGEEEEEEEEDPESP
PKAVKRPAATKKAGQAKKKKLDKEDESSEEDSPTKKGKGAGRGRKPAAKK
Mass spectrometry peptides are highlighted in yellow in the full sequence

of searches.
_______________________________________________________________________________________________________________________________________________________________
D) Sequence alignment of HsMobLak and Xenopus MobLak
Sequence Source
gi|3342738|gb|AAC27672.1| Homo sapiens MobLak
gi|148224716|ref|NP_001089671 Xenopus laevis MobLak
CLUSTAL W (1.83) multiple sequence alignment
gi|3342738|gb|AAC27672.1| KSRRAGVTKMSNPFLKQVFNKDKTFRPKRKFEPGTQRFELHKKAQASLNA 50
gi|148224716|ref|NP_001089671. ---------MSNP-LKQVFNKDRTFRPKRKFEPGTQRFELHKKAQASLNA 40
**** ********:***************************
gi|3342738|gb|AAC27672.1| GLDLRLAVQLPPGEDLNDWVAVHVVDFFNRVNLIYGTISDGCTEQSCPVM 100

gi|148224716|ref|NP_001089671. GLDLKLAVQLPHGEDLNDWVAVHVVDFFNRINLIYGTISDSCTEQSCPVM 90
****:****** ******************:*********.*********
gi|3342738|gb|AAC27672.1| SGGPKYEYRWQDEHKFRKPTALSAPRYMDLLMDWIEAQINNEDLFPTNVG 150

gi|148224716|ref|NP_001089671. SGGPKYEYRWQDDNRYRKPTALSAPKYMNLLMDWIEVQINNEGIFPTNVG 140
************::::*********:**:*******.*****.:******
gi|3342738|gb|AAC27672.1| TPFPKNFLQTVRKILSRLFRVFVHVYIHHFDRIAQMGSEAHVNTCYKHFY 200

gi|148224716|ref|NP_001089671. TPFPKNFLQVVKKILSRLFRVFVHVYIHHFERIIHMGAEAHVNTCYKHFY 190
*********.*:******************:** :**:************
gi|3342738|gb|AAC27672.1| YFVKEFGLIDTKELEPLVRGLGAEGVRNHQVRHLEPPGEGPPSRALKELH 250

gi|148224716|ref|NP_001089671. YFVTELNLIDTKELEPLK------------------------EMTLRMCH 216
***.*:.********** . :*: *
gi|3342738|gb|AAC27672.1| EIRNCLMKCISLYLEDEAQTPTPLSPPGLGMSPAARPRSFP 291

gi|148224716|ref|NP_001089671. -----------------------------------------
A Summary of Mass Spectrometry Results
For each Mob protein, protein bands from polyacrylamide gels that were considered as good
candidates for frog Mob-interacting proteins were sent to mass spectrometry analysis. In MS
analysis, the peptide sequences of these bands were determined. With mascot searches, proteins in
which these sequences existed, were found. The mascot searches for HsPhocein, HsMob2 and
HsMobLak showed many similarities. Mascot searches revealed a high number of sticky proteins for
each of the Mob proteins. Keratin, glutathione-s-transferase, ribosomal proteins, intermediate
filaments, as well as heat shock proteins were the most common. The number of heat shock proteins
were particularly high for HsPhocein and low for the other two Mobs.
Table 1. A Summary of Mascot search results for HsMob2, HsPhocein and HsMobLak. Search
results were obtained by isolating possible Xenopus laevis Mob-interacting proteins from pull down
assays and sending them to mass spectrometry analysis. The number of each type of interacting
protein from Mascot searches for each Mob protein is presented.
_________________________________________________________________________________
Possible Mob- Interacting Proteins HsPhocein HsMob2 HsMobLak

from Mascot Search Results
Total Searches: 38 Total Searches: 91 Total Searches: 11
Glutatione-s-transferase 4 8 3
Ribosomal Proteins 2 30 1
Keratin 6 12 1
Intermediate Filaments 9 10 0
Heat Shock Proteins 8 1 0
Other sticky proteins 9 30 6
Discussion
I. Preparation of Pull Down Assay Bait Proteins and Controls.
A. A discussion of the preparation of GST-fusion bait proteins (GBP) and the GST-control
GST-HsPhocein and GST-HsMob2 were used as bait proteins during pull down assays in this
study. The control used for experiments involving GST-fusion baits proteins (GBP) was GST
alone. The over-expression and purification of GBPs by affinity chromatography was successful.
As presented in results, SDS-PAGE purification gels of GBPs revealed two dominating bands at
approximately 54kDa and 28kDa in various sample lanes (Fig. 1A, 2A and 3A). Both HsPhocein
and HsMob2 are approximately 26kDa, but when fused with the 28kDa GST tag, the molecular
weight is 54kDa. The presence of the 54kDa bands in bacterial lysates and total soluble fractions
are indications that the GST-fusion proteins were expressed (Fig. 1A, 2A and 3A). The decreases
in size and intensity, or in some cases, the disappearance of these bands in the supernatant and
wash flow throughs demonstrate that GST-fusions had bound onto glutathione columns. The
intense 54kDa band in GST-fusion column resins further confirm that the bait protein had bound
onto glutathione (Fig. 1A, 2A and 3A). The existence of 28kDa bands in purification gels
illustrates the activity of bacterial proteases, resulting in the cleavage of the GST-tag from fusion
bait proteins (Fig. 1A, 2A and 3A). Smaller bands below the 54kDa and 28kDa bands represent
other fusion bait breakdown products (Fig. 1A, 2A and 3A). Other protein bands that span the
sample lanes of the bacterial lysate, total soluble fraction and supernatant flow throughs indicate
the presence of other bacterial proteins aside from the GST and GST-fusions (Fig. 1A, 2A and
3A). The GST-control purification gels also showed similar gel patterns with the 28kDa band in
the bacterial crude lysates, total soluble fractions, flow throughs and resins (Fig. 2B and 3A).
Therefore, the GST-control was successfully expressed, purified and had bound onto glutathione
columns.
B. A discussion of the preparation of 6xHis-HsMobLak bait protein (HBP) and 6xHis-B2Nt
In this study, the 6xHis-B2Nt fusion was used as a control in a pull down assay involving the
6xHis-HsMobLak bait protein (HBP). 6xHis-HsMobLak and 6xHis-B2Nt have molecular weights of
approximately 26kDa and 13kDa, respectively. The presence of the 26kDa or 13kDa bands in bacterial
lysates, and total soluble fractions on the SDS-PAGE purification gel can be observed in results, and
indicate that the 6xHis-fusion proteins were expressed (Fig. 5A). The reduction in size and intensity, or
disappearance (in the case of 6xHis-B2Nt) of these bands in the supernatant and wash flow throughs
show that 6xHis-fusion proteins had bound onto Ni-NTA columns successfully (Fig. 5A). This is also
confirmed by the existence of these bands at high intensity in respective column resins (Fig. 5A).
Other bands, aside from the 26kDa and 13kDa bands on the purification gel indicate the presence of
other bacterial proteins (Fig. 5A).
II. Crosslinking of GST-fusion Bait Proteins to Glutathione Agarose Columns
A. The logic behind crosslinking
GBPs bind to glutathione columns through non-covalent interactions between the GST and
glutathione (GST substrate). Chaotropic agents cause disruptions in three dimensional structures of
proteins, and interfere with stabilizations of intramolecular interactions mediated through non-
covalent interactions. In this study, 6M guanidinium hydrochloride (GuHCl) was used to elute
Xenopus laevis prey proteins off GBP columns. Since the interaction of GST with glutathione is non-
covalent, GuHCl would also strip GBPs off columns. As a result, both GBPs and Xenopus laevis prey
proteins would be eluted and appear on SDS-PAGE gels. The presence of GST-fusions and its
proteolytic breakdown products create background on gels and make it difficult to make comparisons
with GST-controls, leading to difficulties in isolating prey protein bands for mass spectrometry
analysis. Crosslinking the GST and GST-fusions onto glutathione columns will allow selective elution
of prey protein from GST-control and GST-fusion columns during pull down assays.
B. Crosslinking of GBP and GST to glutathione, SDS-PAGE gel analysis and troubleshooting.
In this particular study, GST-HsPhocein, GST-HsMob2 and GST were crosslinked to glutathione
columns with dimethyl pimelimidate (DMP). DMP is a homobifunctional crosslinker that possesses
two imidoester groups and reacts with primary amines of GST and glutathione, to covalently link the
two and in effect, immobilize GBPs/GST onto glutathione columns. In theory, if not crosslinked to
glutathione, once placed in SDS-PAGE sample buffer, non-covalent interactions between GBPs/GST
and glutathione should be disrupted, allowing them to be released into solution and create bands at
28kDa and 54kDa on SDS-PAGE gels, respectively. This is demonstrated by HsPhocein, HsMob2 and
GST-control crosslinking gels presented in results (Fig. ). In contrast, if 100% crosslinking
occurred, all GSTs/GBPs should possess covalent interactions with glutathione, and not appear on
SDS-PAGE gels. Realistically, 100% crosslinking efficiency does not occur, and therefore, even after
crosslinking procedures are carried out, uncrosslinked GSTs/GBPs remain on glutathione columns,
lose non-covalent interactions with glutathione in sample buffer, and produce protein bands on SDS-
PAGE gels. Thus, in gels results for all GBPs, crosslinked resins showed bands at 28kDa and 54kDa
(Fig ). It may also be noted that the GST/GBP bands present in crosslinked resin samples are thinner
than those seen in the non-crosslinked resin. The decrease in band thickness represents the fact that
only a portion of the initial non-crosslinked resin reacted with DMP.
In trial one of HsPhocein experiments, the frog egg extract was loaded onto the crosslinked
GST-HsPhocein column. GuHCl eluted samples showed a large band around 28kDa and an intense,
but thinner band at 54kDa (Fig. ). This specifies that GST-HsPhocein was eluted by GuHCl in both
its fusion form, and mostly in the form of GST and other proteolytic breakdown products. Therefore,
the elution of frog prey protein was not selective. The eluted GST-HsPhocein (and its breakdown
products) came from the uncrosslinked GST-HsPhocein that remained on the column after
crosslinking. To troubleshoot against non-selective prey elution, free glutathione solution was used to
elute uncrosslinked GST-HsPhocein off the column after crosslinking. Theoretically, free glutathione
should remove most uncrosslinked GST-HsPhocein from the column. The crosslinked resin after
glutathione wash should reveal very thin fusion protein bands on SDS-PAGE gels. The bands should
also be thinner than the crosslinked resin before glutathione wash, since it should now contain less
uncrosslinked GST-HsPhocein. These expected results are consistent with trial 3 HsPhocein
crosslinking (Fig. ). Similar trends with HsMob2 crosslinking are also observed (Fig. ). Therefore,
in both HsMob2 and trial 3 HsPhocein experiments, GBP columns were favourably crosslinked and
would be appropriate for the use in subsequent pull down assays. An example of unfavourable
crosslinking is represented by trial two of HsPhocein experiments. The glutathione washed crosslinked
resin showed a fusion protein band that was thicker than the crosslinked resin before glutathione wash.
Thus the GST-HsPhocein bait column was not appropriately prepared for pull down assays. In general,
two crosslinking gel patterns need to be observed in order to determine whether or not a GBP column
is fit for the use in pull down assays: 1) a decrease in band thickness from the non-crosslinked to the
crosslinked resin, and 2) the evidence of a decrease in band thickness from the crosslinked resin to the
glutathione washed crosslinked resin. From crosslinking gel analyses, several conclusions can also be
made. First, HsPhocein trial one crosslinking did not include the glutathione wash step and
crosslinking appropriateness could not be determined. However, due to non-selective prey elution seen
in the interacting gel (Fig. ) pull down assay results are invalid. Secondly, HsPhocein trial two
crosslinking was also unsuccessful, and pull down assay results from this trial are also invalid. Finally,
HsMob2 and HsPhocein trial 3 crosslinking experiments were favourable and pull down assay results
from these columns are valid.
C. The GST-Control
In addition to crosslinking errors in trial one of HsPhocein experiments, an insufficient
control was also used. During pull down assay experiments, frog egg extract proteins may also
bind to glutathione, the GST-tag of GST-HsPhocein, and the DMP crosslinker. Once GuHCl is
used to elute potential prey proteins, the glutathione, DMP and GST interacting components
would also be eluted from the GST-HsPhocein column. In trial one, there was no control in
which to compare the GST-HsPhocein eluted samples to, to eliminate non-HsPhocein interacting
frog proteins. The only control used for trial one was the crosslinked resin, which represented all
the contents on the column before the frog egg extract was loaded. This only allowed the
elimination of non-frog protein bands ( i.e bacterial proteins) from the GST-HsPhocein
flocculents and supernatants during SDS-PAGE gel analysis (Fig. ). To provide a solution to
this problem, a glutathione column with crosslinked GST only, was used as a control. The frog
egg extract was run over this column to reduce the amount of GST, DMP or glutathione binding
components, before running it onto the GST-HsPhocein or GST-HsMob2 columns. Frog proteins
were eluted from both the GST-control and GBP columns, and samples were compared on an
SDS-PAGE gel to eliminate any possible GST, DMP or glutathione interacting frog proteins that
might have bound to eluted off the GST-HsPhocein column. The use of the GST-control
maximizes the chance of choosing HsPhocein interacting protein bands for mass spectrometry
analysis. In HsMob2 and trial 3 HsPhocein experiments, eluted samples showed bands that were
different from both the crosslinked resin control, as well as the GST-controls. These bands were
isolated for mass spectrometry analysis as strong candidates for HsMob2 or HsPhocein
interacting frog proteins (Fig. ). Although both the glutathione wash step and the GST-
control was used in trial 2 HsPhocein experiments, the failure in crosslinking caused large
amounts of unwanted GST and GST-HsPhocein to be eluted along with frog prey proteins,
producing background and smearing on gels, and making it difficult to isolate any prey protein
bands (Fig. ). The bands produced from the supernatant and flocculents of the GST-
HsPhocein column were mostly identical to the crosslinked resin control as well, and indicated
no signs of frog egg proteins.
III. Using 6xHis-fusion proteins for Pull Down Assays
In the pull down assay experiment with 6xHis-HsMobLak, problems which were experienced
during crosslinking with the GST-tagged proteins were not encountered. The Ni-NTA column has high
affinity for the tandem histidine tag and is resistant to denaturing and chaotropic agents. Therefore, no
crosslinking procedures were required to immobilize the HsMobLak bait protein. Furthermore, a
different control, another histidine tagged protein, B2Nt (a part of cyclin B), was used as the control.
The choice of this control involved a huge assumption where it was assumed that B2Nt would not
interact with any frog proteins that may interact with HsMobLak. If B2Nt did interact with HsMobLak
interacting frog proteins, running the frog extract through the B2Nt column would eliminate most of
the HsMobLak interacting proteins. Thus, when the extract flow through from the B2Nt column is
loaded onto the HsMobLak column, there would be almost no HsMobLak interacting frog proteins left
to bind to HsMobLak. Furthermore, the choice of magnesium chloride and urea as eluting reagents,
similar to GuHCl was arbitrary, since the nature of interactions between Mob proteins and frog
proteins were unknown.
IV. A Discussion of Mass Spectrometry Results of Possible Mob-interacting Proteins
A. A Brief Overview
For each of the Mob proteins, strong candidates of frog Mob-interacting protein bands
were chosen for mass spectrometry analysis from polyacrylamide gels. The peptide sequences of
these bands were determined by mass spectrometry analysis. These peptide sequences were
placed in Mascot searches to find proteins whose full sequences contained these peptide
sequences. For all Mobs, these searches contained a large number of sticky proteins such as
isomerases, lipid transport proteins, reductases and such. A complete list of Mascot searches for
each Mob can be found in the Appendix. Keratin complexes from buffer and apparatus
contaminations during experiments were also found in searches. In addition, ribosomal proteins
that are involved in celluar processes of translation contributed to a large portion of mascot
searches. In the preparation of frog egg extracts, cells were subject to environmental stress
conditions. This would cause the expression of heat shock proteins. As expected, heat shock
proteins also appeared in mascot searches. However, they were particularly high in number for
HsPhocein as compared to HsMob2 and HsMobLak for reasons unknown. Furthermore,
intermediate filaments which are usually located in the cytosol of cells were also found in mascot
searches. Searches also revealed GST as an eluted component for GST-HsPhocein and GST-
HsMob2 experiments. The presence of GST provides information on what needs to be improved
in experiments. In this study, due to less than 100% crosslinking, it is inevitable that GST-fusion
proteins were eluted in its fusion form, as well as GST and other breakdown products. In trial 3
HsPhocein and HsMob2 experiments, bands around the 20kDa to 30kDa range were isolated for
MS analysis. Thus, it is very possible that these bands contained GST. Another explanation for
the presence of GST is that during the course of experiments with GST-fusions, the fact that
GST can form homodimers was not considered. This means that GST-fusion proteins can bind to
another GST-fusion protein that is crosslinked onto glutathione columns (Fig. ). Thus, when
free glutathione was used to eluted uncrosslinked GST-fusion proteins off glutathione, the
dimerized GST-fusions would not get eluted, since they are not bound onto glutathione itself. As
a result, when eluting with GuHCl, these GST-fusion proteins would be eluted and create GST
breakdown products at 28kDa. In order to reduce the chance of choosing GST bands, the eluted
samples from GST-HsMob2 and GST-HsPhocein columns can be passed through a glutathione-
only column to allow all the GST to bind before running samples on SDS-PAGE gels.
Interestingly, although 6xHis-HsMobLak experiments did not involve the GST-tag, glutathione-
s-transferase also showed up in mascot results. An explanation for this is that GST in Xenopus
laevis may interact with HsMobLak. Of course, this can also be an alternate explanation for the
GST found in GST-HsPhocein and GST-HsMob2 searches.
B. A Discussion of MS results unique to HsPhocein
Polyacrylamide protein bands chosen as good candidates for frog HsPhocein-interacting
proteins revealed three appealing peptide sequences. The peptide sequences were
K.DLQSGIHIK.K, R.IFSHAYFHHR.Q and K.YSFLQFDPAPR.R and were contained in the full
sequences of calpain 3, hypothetical protein, and protein phosphatise 2A, respectively. The
scores for these mascot searches were 37(calpain), 45(hypothetical protein) and 98 (PP2A).
The Hypothetical Protein
The full sequence of the hypothetical protein was found to be identical to the sequence of
Xenopus laevis Phocein. When aligned in a sequence alignment with HsPhocein, it obtained a
score of 97 percent similarity (Fig 3E). The sequence alignment also indicated that the majority
of the amino acids between the two sequences were conserved (Fig. ). Both HsPhocein and
Xenopus laevis Phocein contain the peptide sequence R.IFSHAYFHHR.Q (Fig. 3E). From these
results, two possible solutions can be identified. First, since both the frog and human Phoceins
contain the peptide sequence in their full sequences, it is difficult to determine where the peptide
came from; from HsPhocein or Xenopus laevis. Although the peptide came from a protein band
in the GST-HsPhocein eluted samples, it is possible that unknown proteases in Xenopus laevis
could cleave the HsPhocein off of its GST tag which is crosslinked to the glutathione column,
giving rise to the HsPhocein band. In this case, it can not be concluded that a Xenopus laevis
phocein interacts with HsPhocein. In the other case, if no proteases cleaved the HsPhocein off of
the GST, then it is possible that Xenopus laveis Phocein interacts with HsPhocein. When placed
into sample buffer, protein bands of Xenopus laevis Phocein arise. The uncertainty in the results
would require future protein-protein interaction confirmational experiments with yeast two-
hybrid, co-immunoprecipitation and western blotting or gel filtration chromatography.
Protein Phosphatase 2A
Another possible frog HsPhocein interacting protein was protein phosphatase 2A (PP2A).
PP2A is a serine/threonine phosphatase that consists of three different subunits including the A
structural subunit, the regulatory B subunit and the catalytic C subunit (Moreno et al., 2001). The
phosphatase has been known to be involved in many cellular processes dealing with
development, viral transformation, neuronal signalling, as well as cell cycle regulation. The A
subunit is a member of the HEAT repeat protein family which acts as a scaffolding unit for the
formation of the PP2A heterotrimeric complex. When interacted with the catalytic subunit, it can
alter its enzymatic abilities, even in the absence of the B subunit. A and C subunits have shown
to be conserved at the amino acid level throughout eukaryotes, while B subunits are varied. A
recent study has showed that SG2NA (S-phase/G-Phase nuclear autoantigen) and striatin form
stable complexes with the A and C heterodimer of PP2A. SG2NA and striatin are part of the
striatin family of proteins which contain multiple protein-protein interaction domains including
the caveolin binding domain, a potential coiled-coil structure, a calmodulin binding domain, a
membrane binding domain and a WD repeat domain. Therefore, both SG2NA and striatin can
function as scaffolding proteins which act to assemble a variety of proteins into complex with
PP2A (A/C heterodimer). Through immunopurification, the mammalian class II homolog of the
yeast protein Mob1 has been found to be a member of striatin-PP2A and SG2NA-PP2A
complexes. This could be a possible explanation for the mascot search result of PP2A as a frog
HsPhocein interacting protein.
In S. cerevisiae, Mob1 is required for mitotic exit, maintaining cell ploidy and mitotic
spindle body duplication. In S. pombe, Mob1 is localized to the spindle pole bodies during the
cell cycle, as well as to the medial ring in late mitosis and is involved in cytokinesis. As
explained earlier, Mob1 interacts with a Dbf2-like kinase which indirectly causes the release of
cdc14, a phosphatase that drives mitotic exit through dephosphorylation and inactivaiton of
Cdks. Cdc14p targets late mitotic substrates, such as Cdh1p, sic1p and Swi5p; Cdk substrates
that regulate mitotic cyclin degradation, Cdk inactivation, and G1 gene transcription,
respectively. Proteins in the FEAR pathway control the early release of cdc14p and LTE1
encodes the guanine nucleotide exchange factor for Tem1, a GTP binding protein responsible for
initiating the MEN pathway. Recent studies have shown that a deletion in PP2A can allow the
enhancement of suppression of mutations in mitotic exit genes including slk19, lte1, and tem1.
Thus PP2A plays a negative role in mitotic exit. It is a possibility that Mob1 may also interact
with PP2A, to inhibit it and cause the suppression of FEAR gene mutations, initiate cdc14
release and cause mitotic exit. In yeast, Mob1 (phocein) interacts with a Dbf2-like kinase which
indirectly causes the release of cdc14. It is also possible that Xenopus laevis PP2A may cause a
dephosphorylation in the same site that Dbf2 phosphorylates, inhibiting the release of cdc14 and
thus inhibiting mitotic exit. However, why Dbf2-like kinases were not seen in pull down assays
and mascot searches is unclear. The mascot search of PP2A as a frog HsPhocein interacting
protein seems likely, especially with its high score of 98, compared to all other searches. Further
immunoprecipitation experiments need to be conducted to confirm the interaction between
PP2A and frog phocein before any definite characterizations of the nature of the interaction can
be made.
C. A Discussion of HsMob2 Mass Spectrometry Results

The polyacrylamide protein bands of possible HsMob2 interacting frog proteins sent to
mass spectrometry analysis yielded three interesting peptide sequences: K.LVTDEDVFPTK.Y,
-.MNVLQKNK and K.DTEGLVQEMNELR.L. The peptide sequences were found by mascot
searches to be contained in the full sequences of Xenopus laevis Mob2, bone morphogenetic
protein 7 and liprin beta 1, respectively.
The Xenopus laevis Mob2protein
The score for this mascot search was 55 and was considered as significant according to
Mowse probability distributions. The mascot search initially identified this search as an unknown
protein that contained the region of mob1_phocein. This sequence, containing the mass
spectrometry peptide was aligned in a sequence alignment with HsPhocein, as well as frog
phoceins (Fig. 4F). The alignment r between the human and frog Phocein sequences resulted in
an identity score of 96, 97 and 98 percent identical. However, when aligned with the unknown
protein sequence, the score was 13. Thus, the unknown protein sequence provided by the mascot
search was most likely not phocein. Furthermore, the K.LVTDEDVFPTK.Y peptide was not
represented in any of the phocein sequences. It was later discovered that the unknown protein
sequence was actually Mob2. This suggested that Xenopus laevis Mob2 may interact with
HsMob2. However, since Mob2 sequences between frog and human are virtually identical, it is
difficult to determine where the Mob2 in the eluted samples came from. As with HsPhocein
experiments, it is of course possible that the HsMob2 was cleaved off by frog proteases from the
GST tag on the bait column and was eluted with frog prey protein. In this case, it was HsMob2
that was eluted and not a frog interacting Mob2. Analogous to the situation with HsPhocein
experiments, western blots, immunoprecipitation, yeast two hybrid and gel filtration
chromatography could be conducted to confirm these interactions and further characterize them.
The Liprin beta 1 Protein

LAR, the leukocyte common antigen-related subfamily of receptor protein tyrosine
phosphatases have shown to have functions in neural development and various pathologies such
as diabetes and cancer. Their extracellular domain is similar to the super immunoglobulin family
and allows potential interactions with a complicated network of membrane-associated proteins
involved in cell-cell and cell-extracellular matrix interactions. More specifically, LAR-RPTPs
are known to be involved in adhesion signalling as adhesion molecules and transducer
extracellular signals to intracellular signalling via PTP domains. LAR-interacting proteins, such
as liprins contain SAM and coiled-coil domains that are responsible for many protein to protein
interactions. Once interacted, the LAR-liprin complex localizes to focal adhesions where actin
remodelling of the cytoskeleton into stress fibers occurs. Lipins act to recruit LAR-RPTPs within
the vicinity of its substrates which together with LAR-RPTPs allow various remodelling of the
cytoskeleton. Liprins are therefore involved in cell cycle regulation. In this study, mascot
searches identified Liprins as possible HsMob2 interacting frog proteins. It is possible that liprins
interact with HsMob2 to recruit it to areas where remodelling of cytoskeleton takes place and
could be an explanation of the appearance of liprin in mascot results. There has been no other
studies on the interaction of liprins and Mob proteins. Again, these hypotheses need to be tested
by yeast two-hybrid or immunopreciptation experiments. Furthermore, the score for the liprin
result was 35, just above the 32 cut off from the mowse probability distribution. Although
considered as a significant result, the score is low compared to the other mascot results. Thus, it
is also likely that HsMob2 and liprin do not even interact. Results are inconclusive and the result
was unexpected.
The Bone Morphogenetic Protein 7(BMP7)
Recent studies from Yan and Chen (2007) have shown that the bone morphogenetic
protein 7 is a target of p53 and together regulate cell survival functions. Their studies showed
that the knockdown of BMP7 inhibited the proliferation of p53 deficient breast cancer cells.
Furthermore, BMP7 plays roles in the maintenance of p53 deficient cells and that cancer patients
without p53 may be benefited by targeted repression of BMP7. Similar to p53, mammalian Mob
proteins may also act as tumor suppressors called Mats which colocalizes with Lats kinase (
large tumour suppressor) and cycline E proteins at the centrosome for mitotic control. It is
possible that BMP7 may interact with Mob2 in Xenopus laevis, similar to p53 and regulate cell
survival functions. Thus, the mascot search of BMP7 is not an unexpected result, but again,
interactions need to be confirmed with yeast two hybrid and immunoprecipitation experiments.
The score of 45 for this mascot search is also well above 32, but is lower than many other mascot
searches.
D. A Discussion of HsMobLak Mass Spectrometry Results
The polyacrylamide protein bands of possible frog HsMobLak interacting proteins sent for mass
spectrometry resulted in two significant peptide sequences: K.TVALWDLR.N, and
K.AKDEFHIVEIVTQEEGK.E which were contained in full sequences of two proteins provided
by mascot searches. The proteins were the retinoblastoma protein 7, and nucleoplasmin,
respectively
The Retinoblastoma Binding Protein 7
RB, the retinoblastoma gene is one of the most studied tumour suppressor genes. The
loss or inactivation of this gene results in the loss of cell proliferation control. Retinoblastoma
binding proteins, such as RB protein 7 contain the Rb-binding motif, LXCXE. RBBP7 is
normally a nuclear protein expressed ubiquitously and is amoung the highly conserved subfamily
of WD-repeat proteins. It binds directly to retinoblastoma protein to regulate cell proliferation.
RBBP7 is also found in histone deacetylase complexes such as the msin3 co-repressor complex.
Furthermore, it appears in chromatin assembly complexes and interacts with BRCA-1 tumour
suppressor genes and regulate proliferation and differentiation of cells. As suggested by mascot
results, frog RBBP7 interacts with HsMobLak and the complex may work analogously to
RBBP7-RB. Although mass spectrometry results suggest the interaction between RBBP7 and
HsMobLak, these interactions need to be confirmed with future experiments with yeast two
hybrid systems and immunoprecipitation methods.
The Nucleoplasmin Protein
The most abundant protein in the nucleus of Xenopus laevis oocytes is nucleoplasmin
which has a molecular weight of 30kDa, and forms stable pentamers in vivo. In oocytes, the
pentamer is complex to histones, allowing them to assemble histones onto nucleosomes when
DNA is present. Burglin et al. (1986) have shown that the amino acid sequence of nucleoplasmin
has a very hydrophilic carboxyl terminus and contains a length of 12 glutamic acid residues and
is fit for its function in promoting chromatin assembly. Nucleoplasmin acts as a histone
chaperone involved in nucelosome assembly, chromatin decondensation at fertilization, as well
as apoptosis. These activities are carried out through the interaction with histones. This
interaction is regulated by phosphorylation. Thus, nucleoplasmins also play important roles in
the cell cycle. Mass spectrometry results indicate that nucleoplasmins interact with HsMobLak.
Again, in order to understand the function of this interaction, the interactions have to be
confirmed by yeast two hybrid and immunoprecipiation experiments.
General Concerns and Future Directions
In all pull down assay experiments, homo sapien bait proteins were used. Mass spectrometry
results also revealed possible Xenopus laevis proteins that interacted with each of the bait proteins.
However, it can be assumed that these proteins also interact with Xenopus laevis Mob proteins. This is
because, as sequence alignments between Xenopus laevis and homo sapien Mob proteins show, these
proteins are virtually identical, obtaining high identity scores (Fig. ).
Mass spectrometry results indentified many Xenopus laevis proteins that may possibly interact
with Mob proteins. Since there have been limited studies on the interactions of Mob proteins with
other proteins, it was not known what to expect. Several proteins were expected to show up in mass
spectrometry results, and were based on several mammalian and yeast mob-interacting proteins in. For
instance, Ndr kinases, or Dbf2-like kinases did not appear. These proteins may also be in Xenopus
laevis and interact with Mob proteins, but did not appear in results. This created some doubt in
determining to whether or not pull down assays were all that successful. This suggested that not all
Mob-interacting proteins were pulled down. Although this is the case mass spectrometry results can
not be eliminated and may possibly interact with Mob proteins. To be sure, the interactions of Mob-
proteins and its interacting proteins must be confirmed by gel filtration chromatography to allow the
elution of complexes by sizes, yeast two hybrid detections and immunoprecipitation. Nonetheless, the
results of this study provide a foundation and focus in which Mob-interacting protein interactions can
be studied.
Figure 6: A) A theoretical diagram of the components of a GST-HsPhocein saturated glutathione column crosslinked with
dimethyl pimelimidate. The diagram shows a single agarose bead crosslinked to a glutathione which is crosslinked to the GST-
HsPhocein or HsMob2 that it binds. The GST binds the glutathione with its GST binding site between its N-terminal and C-
terminal domain. The figure also shows the homodimerization of GST in A) GST-HsPhocein or GST-HsMob2 columns and in B)
GST-control columns.
_________________________________________________________________________________________________________________________________________
A B
) )
Appendix
Table I. Reagent Recipes for preparation of bait protein cell lysates.
Reagents HsMob-Lak HsPhocein HsMob2

Lysis Buffer 500mM NaCl( ), 50mM 1X phosphate 140mM NaCl,
NaH2PO4( ) 5mM buffered saline, 2.7mM KCl(
imidazole ( ), 0.5% 1% Triton X- ), 10mM Na2HPO4 (
Triton X-100(Sigma-Aldrich, 100 at pH 7.2 ), 1.8mM KH2PO4 (
St Louis, MO) , 5% ), 1% Triton X-100
glycerol ( ) at at pH 7.3
pH 7.2
Benzamidine ( 200ul of 1.0 M stock 300ul of 1.0M 100ul of 1.0M stock
) stock
PMSF (Sigma- 200ul of 0.2M stock 300ul of 0.2M 100ul of 0.1M stock
Aldrich, St Louis, stock
MO)
Dithiothreitol ( N/A 150ul of 1M N/A
) stock

Biochemistry 528 Paper 11

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Biochemistry 528 Paper 11

Hochgeladen von

Copyright:

Verfügbare Formate

Introduction

prophase, prometaphase, metaphase, anaphase, and telophase. Once mitosis is completed,

phase of the cycle is regulated in a cyclin-Cdk (cyclin-dependent kinase) dependent manner,

for mitotic exit, as phosphorylated cyclin-Cdk complexes need to be dephosphorylated to

inactivation of Cdks, promotion of cytokinesis, activation of G1 gene transcription, and

formation of pre-replication complexes (Stoepel et al., 2005). Regulatory proteins involved in

Bub2p-Bfa1p, and leads to the accumulation of GTP-Tem1p, activating Cdc15, which

transcription, respectively (Yoshida and Toh-e, 2001).

In S. cerevisiae, Mob2 exists as a Mob1-like component of the Ram (regulation of Ace2p

Mob1p-Dbf2p in MEN (Weiss et al., 2002).

The septation initiation network (SIN) in fission yeast (Schizosaccharomyces pombe) is

(Spg1p), a GTPase-activating protein complex (Cdc16p-Byr4p), a SPB anchoring complex

activation of Mob1p-Sid2p via Cdc7p, and sid1p-cdc14p allow localization of Mob1p-Sid2p to

Furthermore, other possible Mob-interacting proteins in higher eukaryotics have yet to be

through a variety of methods including: two-hybrid, affinity capture, co-crystallization, and

can be applied to other similar vertebrates, as well as humans.

Material and Materials

Over-expression of Mob Bait Proteins

and stored at -80oC until purification.

streaked onto a LB plate containing 75ug/ml ampicillin. Inoculation, dilutions, expression,

unpublished) was grown on a LB plate containing 30ug/ml kanamycin, (company name

Affinity Purification of Mob Bait Proteins

A. Preparation of Appropriate Affinity Columns For Bait Proteins

appendix for wash buffer and lysis buffer recipes.

B. Preparation of Cell Lysate

(Aroian Lab Protocols)

To immobilize GST-fusion proteins onto glutathione columns, dimethyl pimelimidate

Capturing Potential Mob-interacting Proteins from Xenopus Laevis

________ apparatus. A detailed procedure can be found in the appendix.

Three trials of experiments were conducted to find possible HsPhocein-interacting

proteins from Xenopus laevis.

HsPhocein Interacting Experiments Trial 1

A)HsPhocein purification SDS-PAGE gel

Gel Run Time: 1 hour and 15 minutes

Gel Run Time: 1 hour and 5 minutes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 C) HsPhocein-interacting protein SDS-PAGE gel

Gel Run Time: 1 hour and 5 minutes

through show no signs of the same bands (Fig.2B).

28kDa in the GST-HsPhocein resin samples (Fig. 2C).

glutathione washed crosslinked resin (blue boxed, Fig. 2E).

15 Gel Run Time: 1 hour and 30 minutes

1 2 3 4 5 6 7 8 9 10 C) GST-control and HsPhocein Crosslinking SDS-PAGE

15 Gel Run Time: 1 hour and 15 minutes

97 similar (Fig. 3E).

1 2 3 4 5 6 7 8 9 10 11 12 A) GST-HsPhocein and GST-control purification SDS-

200 1 2 3 4 5 6 7 8 9 10 11 12 C) GST-HsPhocein interacting SDS-PAGE gel of GnCl

D) Two important Mascot searches of interest from mass spectrometry analysis

Full sequence of hypothetical protein given by search 2

SeqA Name Len(aa) SeqB Name Len(aa) Score

gi|148228740|ref|NP_001088946. CSNIDKILDPPEGQDEGVWKYEHLRQFCLELNGLAVKLQTECHPDTCTQM 100

gi|148228740|ref|NP_001088946. TATEQWIFLCAAHKTPKECPAIDYTRHTLDGAACLLNSNKYFPSRVSIKE 150

gi|148228740|ref|NP_001088946. SSVAKLGSVCRRIYRIFSHAYFHHRQIFDEYENETFLCHRFTKFVMKYNL 200

gi|148228740|ref|NP_001088946. MSKDNLIVPILEEEVQNSVAGESEA 225

crosslinked and washed with glutathione (Fig. 4A).

28kDa than the glutathione washed crosslinked resin (Fig. 4B).

200 1 2 3 4 5 6 7 8 A) GST-control SDS-PAGE purification gel

B) GST-HsMob2 SDS-PAGE purification gel

Gel Run Time: 1 hour and 36 minutes

1 2 3 4 5 6 200 7 8 9 10 11 12 C) GST-HsMob2 interacting SDS-PAGE gel 1 of GnCl

Gel Run Time: 1 hour and 10 minutes

D) GST-HsMob2 interacting SDS-PAGE gel 2 of GnCl eluted

SeqA Name Len(aa) SeqB Name Len(aa) Score

gi|62857563|ref|NP_001017210.1 CSNIDRILDPPEGQDEGVWKYEHLRQFCLELNGLAVKLQTECHPDTCTQM 100