1. Production
and
binding
of
D4
protein
Bree
Drda
Purpose
C.
botulinium
C2II
domain
four
(D4)
is
a
toxin
binding
domain
protein
with
a
general
specificity
towards
eukaryotic
cell
surface
glycosylation
patterns
which
promotes
cellular
recognition
and
endocytosis
of
the
toxin.
It
has
not
yet
been
characterized
by
crystallography
or
used
in
a
biomedical
application
as
a
binding
domain.
Other
research
groups
1,2,3
have
also
investigated
this
protein
and
this
project
is
a
continuation
of
their
work.
The
purpose
of
this
report
is
to
describe
the
methods
and
results
of
producing
D4
protein
and
binding
it
to
the
endosomes
of
N2A
cells.
DNA
electrophoresis,
SDS-­‐PAGE,
and
confocal
microscopy
were
used.
Methods
Cell
line
and
expression:
The
DNA
for
D4
was
amplified
from
the
vector
BDV/pGex-­‐2T
by
PCR
with
a
5’
BAMHI-­‐glycine
extension
and
a
3’
ECORI
extension
using
primers
GCGGGATCCGGTCGTAAGGAAAACATCTCATCGATCAACATCATCAACG
and
CCGGAATTCTTAGATAATCAGTTTATCCAGTTCAATCAGAAACACGCCCGACAGAC.
The
vector
BDV/pGEX-­‐2T
was
obtained
from
synthesized
DNA
by
Genscript
with
codon
optimized
for
E.
coli
expression.
This
insert
corresponded
to
amino
acids
593-­‐721
of
PDB
entry
2J42.
The
amplified
fragment
was
ligated
into
the
vector
pGEX-­‐2T
by
directional
cloning
at
the
restriction
sites
BamHI
and
EcoRI
to
make
the
expression
vector
pGEX-­‐2T:D4.
DH5α
was
transformed
to
propagate
the
vector
and
BL21
(DE3)
was
chosen
as
the
expression
host
strain
and
transformed.
The
D4
sequence
was
confirmed
by
Operon
sequencing
services
and
did
not
contain
errors.
A
colony
was
grown
in
3
mL
culture
tubes
with
LB
media
and
100
ug/mL
of
ampicillin.
After
incubating
for
18
hours
at
37°C,
1
mL
of
the
culture
was
added
to
400
mL
of
LB
media
and
grown
until
an
OD
of
0.3
–
0.5
was
reached.
The
culture
was
induced
with
0.5
mM
IPTG
once
it
had
reached
an
OD
of
0.5
–
0.8.
It
was
then
incubated
for
three
hours.
The
cells
were
pelleted
by
centrifuging
the
mixture
at
5,000
rpm,
4°C,
for
10
minutes
using
100
mL
of
culture/pellet.
The
pellets
were
stored
at
-­‐20°C.
Protein
extraction
and
purification:
A
D4
cell
pellet
constituting
100
mL
of
culture
volume
was
resuspended
in
20
mL
of
1
x
PBS
+
1%
Triton
pH
7.5.
The
sample
was
pressed
3
times
at
1000
psig,
(~16,000
psia
cell
pressure)
using
a
French
press.
The
cell
debris
was
pelleted
in
a
centrifuge
at
10,000
rpm,
4°C,
for
20
minutes
and
the
supernatant
containing
the
protein
was
decanted.
This
supernatant
was
then
incubated
with
washed
immobilized
glutathione
resin
(Genscript)
for
one
hour
at
4°C
to
let
the
protein
bind
with
the
resin.
The
supernatant
was
washed
of
excess
non-­‐binding
protein
by
centrifuging
the
resin
with
two
volumes
of
8
mL
of
1
x
PBS
+
1%
Triton.
The
Triton
was
then
removed
by
centrifuging
with
three
volumes
of
1
x
PBS.
All
spins
involving
the
resin
were
done
at
2,000
rpm,
4°C,
for
five
minutes.
The
final
total
volume
of
the
solution
after
the
last
wash
was
then
reduced
to
1
mL,
and
10
units
of
thrombin
were
added
in
order
to
cleave
the
protein
from
the
resin.
The
resin
and
the
protein
dissolved
in
the
supernatant
were
separated
using
a
syringe
plugged
with
glass
wool.
The
protein
elution
was
concentrated
to
~ten
times
the
initial
concentration
using
3K
spin
column
filters
and
stored
at
4°C.

2. Protein
Induction
with
N2A
Cells:
Four
well
plates
on
a
24-­‐well
well
plate
were
seeded
with
100,000
N2A
cells
in
1
mL
of
EMEM
+
10%
FBS
+
1%
Pen
Strep
and
grown
at
37°C
with
5%
CO2.
After
24
hours,
three
of
the
wells
were
inoculated
with
Bacmam
2.0
early
endosome
labeling
kit
(Molecular
Probes)
baculovirus
at
a
concentration
of
50
particles
per
mammalian
cell
(PPC)
and
left
to
incubate
at
37°C
for
24
hours.
D4
protein
labeled
with
Alexa
Fluor
was
added
to
the
well
plates
at
following
concentrations:
0,
1,
5,
and
10
µg/mL.
The
cells
were
then
fixed
with
4%
paraformaldehyde
and
stained
with
DAPI.
The
collagen
coverslips
were
removed
and
mounted
on
glass
slides.
The
cells
were
examined
using
confocal
microscopy
at
60X
zoom.
Results
Protein
purification
gel:
Lanes
7
and
8
show
the
purified
D4
protein
after
it
has
been
cut
from
the
resin.
The
amount
of
material
present
in
lane
5
is
considerably
more
than
lane
9.
This
suggests
that
the
protein
bound
to
the
resin
in
lane
5
has
been
cut
off,
which
is
what
we
see
appearing
in
lanes
7
and
8.
There
are
some
impurities
present
in
lane
7,
which
can
be
seen
more
distinctly
in
lane
6,
which
is
the
concentrated
fraction.
The
purity
of
the
protein
in
lanes
7
and
8
is
estimated
to
be
90%.
Lanes
1 – Ladder
2 – Cell debris pellet
3 – Supernatant
4 – Final wash supernatant
5 – Resin, pre-thrombin
6 – Concentrated elution fraction from 10/12
purification (same cell paste lot)
7 – Elution fraction 1
8 – Elution fraction 2
9 - Resin, post-thrombin
Expected
Masses
kDa
D4/pGex-­‐2T
40
D4
14

3. DNA
gel
of
restriction
digest
of
transformed
DH5a
to
screen
for
inserted
D4
DNA
This
gel
shows
a
digest
of
the
vector
plus
D4
insert
by
BamHI/EcoRI
restriction.
This
confirms
the
presence
of
the
D4
gene
in
the
appropriate
cloning
site
for
pGEX-­‐2T.
The
expected
masses
of
the
fragments
are
4.9
kb
(pGex-­‐
2T)
and
0.4
kb
(D4).
Screen
for
D4
inserted
in
pGex-­‐2T:
D4
in
DH5α
DNA
gel
1
–
GeneRule
1kb
Plus
DNA
ladder
2
–
colony
4
from
isolate
patch
plate
3
–
colony
5
from
isolate
patch
plate
20000
10000
7000
5000
4000
3000
2000
1500
1000
700
500
400
1
2
3

4. Confocal
microscopy,
60
X
Zoom:
These
figures
show
N2A
cells
that
were
incubated
with
10
µg/mL
D4
protein.
Bacmam
2.0
targets
specifically
endosomes.
The
overlap
of
green
and
red
areas
in
Figure
3
and
Figure
4
shows
the
colocalization
of
Bacmam
2.0
and
D4.
Figure
1:
N2A
cells
incubated
with
Bacmam
2.0
(green),
DAPI
(blue),
and
Alexa
Fluor
568
labeled
D4
(red).
a)
Bacmam
2.0
b)
Alexa
Fluor
568
c)
Bacmam
2.0
+
Alexa
Fluor
568
d)
Bacmam
2.0
+
Alexa
Fluor
568
+
DAPI
Figure
2:
Zoom
of
Figure
1c
a)
b)
c)
d)
Figure
3:
Zoom
of
Figure
1d

5.
From
these
images
it
appears
that
there
is
colocalization
between
D4
and
early
endosomes
(Rab5a).
In
figure
1D
there
is
a
lack
of
green
label,
but
it
appears
that
D4
has
still
localized
to
a
similar
location
as
protein
that
has
green
labeling
in
the
same
frame.
Therefore,
we
are
assuming
that
this
protein
is
most
likely
also
colocalized
to
endosomes.
Discussion:
The
confocal
microscope
images
show
cells
with
areas
where
Bacmam
2.0
and
labeled
D4
overlap
(colocalization)
and
are
represented
by
a
yellow
color.
Bacmam
2.0
targets
the
endosomes
of
cells
as
a
GFP/Rab5a
fusion,
bringing
GFP
to
Rab5a
locations.
Rab5a
is
a
protein
that
specifically
localizes
to
early
endosomes.
The
fact
that
the
two
are
present
in
the
same
areas
of
cells
shows
that
D4
is
in
the
same
subcellular
location
as
the
endosomes
of
cells.
Therefore,
it
is
plausible
that
D4
may
be
useful
as
an
endosomal
targeting
moiety
in
a
drug
delivery
application.
Based
on
the
SDS-­‐PAGE
results,
we
can
see
that
this
purification
procedure
produces
D4
protein
at
an
estimated
90%
purity.
Determining
the
structure
of
this
protein
with
crystallography
would
require
~15
mg
of
highly
pure
D4
protein.
This
batch
of
D4
E.
coli
cells
produced
0.5
mg/L.
In
the
future,
we
will
explore
using
a
reactor
in
order
to
produce
more
D4
protein.
Appendix:
D4
construct
sequences
This
corresponds
to
amino
acid
residues
593
to
721
with
the
addition
of
GR
at
the
N-­‐terminus
for
thrombin
cleavage
consensus.
Figure
4:
N2A
cells
incubated
with
Bacmam
2.0
(green),
DAPI
(blue),
and
Alexa
Fluor
568
labeled
D4
(red).
a)
Bacmam
2.0
+
Alexa
Fluor
568
+
DAPI
b)
Alexa
Fluor
568
c)
Bacmam
2.0
d)
Bacmam
2.0
+
Alexa
Fluor
568
a)
c)
b)
d)
Figure
6:
Zoom
of
Figure
4a

6. 1 GRKENISSIN IINDTNFGVE SMTGLSKRIK GNDGIYRAST KSFSFKSKEI 50
51 KYPEGFYRMR FVIQSYEPFT CNFKLFNNLI YSNSFDIGYY DEFFYFYCNG 100
101 SKSFFDISCD IINSINRLSG VFLIELDKLI I 131
DNA sequence confirmed by sequencing
1 GGATCCGGAT TCATGCGTAA GGAAAACATC TCATCGATCA ACATCATCAA 50
51 CGACACGAAC TTCGGCGTGG AAAGTATGAC CGGTCTGTCC AAACGTATTA 100
101 AGGGCAACGA TGGTATCTAT CGCGCGTCAA CCAAATCGTT TAGCTTCAAA 150
151 TCGAAGGAAA TTAAGTACCC GGAAGGTTTT TATCGTATGC GCTTCGTTAT 200
201 CCAGTCTTAT GAACCGTTCA CCTGTAACTT CAAGCTGTTC AACAACCTGA 250
251 TCTACTCTAA CAGTTTCGAC ATCGGCTACT ACGATGAATT TTTCTACTTC 300
301 TACTGCAACG GTTCCAAATC ATTTTTCGAC ATCAGTTGTG ATATCATCAA 350
351 CTCAATCAAC CGTCTGTCGG GCGTGTTTCT GATTGAACTG GATAAACTGA 400
401 TTATCTAAGA ATTC 414
References
1. Schleberg,
C.,
Hochmann,
H.,
Barth,
H.,
Aktories,
K.,
&
Schulz,
G.
E.
(2006)
Structure
and
Action
of
the
Binary
C2
Toxin
from
Clostridium
botulinum.
Journal
of
Molecular
Biology,
364,
705-­‐715.
2. Eckhardt,
M.,
Barth,
H.,
Blöcker,
D.,
&
Aktories,
K.
(2000)
Binding
of
Clostridium
botulinum
C2
Toxin
to
Asparagine-­‐linked
Complex
and
Hybrid
Carbohydrates.
The
Journal
of
Biological
Chemistry,
275(4),
2328-­‐2334.
3. Nagahama,
M.,
et
al.,
Binding
and
Internalization
of
Clostridium
botulinum
C2
Toxin.
Infection
and
Immunity,
2009.
77(11):
p.
5139-­‐5148.