prokaryotic expression of human complement regulator ...1147592/fulltext01.pdf · master thesis...
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INOM EXAMENSARBETE BIOTEKNIK,AVANCERAD NIVÅ, 30 HP
, STOCKHOLM SVERIGE 2017
Prokaryotic expression of human complement regulator factor H domains and their interaction with Pneumococcal surface protein PspC.
NILS LINDSTRÖM
KTHSKOLAN FÖR BIOTEKNOLOGI
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Royal Institute of Technology
Prokaryotic expression of human complement regulator factor H domains and their interaction with Pneumococcal surface protein PspC. Master Thesis Project Report
Nils Lindström 2017-09-21
Abstract
Virulent strains of S. pneumoniae are known to evade the alternative pathway of
complement immunity by means of a surface bound protein called PspC that recruits the
human complement regulator Factor H. Factor H is a self surface marker that inhibits the
activity of the alternative pathway of complement immunity and is comprised of 20
Complement Control Protein (CCP) domains in a “bead on a string” fashion. It has been
concluded that PspC can use two different mechanisms of binding Factor H. The PspC
allele of the TIGR4 strain of S. pneumoniae has been shown to have affinity for the 9th
CCP of Factor H through a “lock and key” mechanism mediated by a critical Tyr90
residue. However, PspC from the D39 strain of S. pneumoniae does not posess Tyr90 and
it hasn’t been conclusively shown which CCP it binds to. In an effort to elucidate this
mechanism, individual CCP domains were expressed as fusion proteins with Maltose
Binding Protein in an E. coli based expression system. The fusion proteins were used in
experiments with recombinant PspC cloned from the BHN_418 strain of S. pneumoniae
which is homologous to that of D39 PspC. Affinity interactions were investigated with a
pulldown assay, copurification, microscale thermophoresis and ligand tracer assays. The
results are inconclusive. The recombinant PspC is shown to bind full length Factor H but
not any of the individual CCP-MBP fusion proteins, most likely due to the CCPs failing
to achieve proper tertiary structure during expression.
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Master Thesis Report Royal Institute of Technology Nils Lindström
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Introduction Community acquired bacterial pneumonia is
still one of the most deadly infectious diseases
in the world and in almost 50% of cases
Streptococcus pneumoniae can be isolated as
the causative pathogen.- 1. S. pneumoniae is a
gram positive, facultative anaerobic and
commensal bacterium that often colonizes the
mucosal surfaces of the respiratory tract and
nasopharyngeal space.2 It is responsible for 30-
60% of acute otitis media worldwide 3 as well
as meningitis, sinusitis, sepsis and other acute
infections in immunocompromised individuals
like HIV-patients 4. Estimates from 2009
indicate that circa 800 000 children under the
age of five die each year from S. pneumoniae
infections 5. An important virulence factor of S.
pneumoniae is Pneumococcal Surface Protein C
(PspC), also known as SpsA, Hic or CbpA.
Apart from acting as an adhesion molecule It is
known to recruit the human complement
regulator factor H, (fH) on the bacterial surface
in order to evade complement deposition. fH is
a 155kDa protein comprised of 20 domains
known varyingly as complement control
proteins (CCPs), Short Consensus Repeats or
sushi domains. It has an unusual structure where
the 20 CCP domains are connected with short
peptide linkers in a “bead on a string” fashion,
where the different domains of the protein have
different functions in vivo.6 S. pneumoniae
utilizes the affinity for fH to mediate adhesion
to host epithelial cells and to circumvent the
complement system of immunity. 7 The latter
property is enabled because of the regulatory
functionality of fH on the alternative pathway
of the complement immune system. The
alternative pathway of complement immunity is
in a state of constant equilibrium between a
positive feedback loop that generates
opsonizing C3b and the repressive effect that fH
has on this loop. Complement protein C3 is
spontaneously cleaved in small amounts in the
bloodstream and when this occurs in close
proximity to a cell surface the resulting
fragment C3b is covalently bound to it by its
revealed nucleophilic thioester domain. Surface
bound C3b recruits factor B to produce the C3
convertase or C3bBb complex which acts as a
potent catalyst for further cleavage of C3. This
action constitutes a positive feedback loop
which will eventually result in all nearby
surfaces being opsonized by C3b which leads to
the formation of the Membrane attack complex,
cell lysis and phagocytosis. To prevent this to
occur on self surfaces fH acts as catalyst for
removing the Factor Bb subunit of the C3-
convertase and form a complex called C3b-
Factor H which possesses affinity for Factor I
which further cleaves the complex to produce
iC3b and C3c which can take no further part in
the amplification loop.8,9
Figure 1 - The alternative pathway of immune system activation with available structure data. CVF-Factor B is homologous
to C3b-Factor B. Graphics by Serruto et.al. 2010 Nature Reviews .8
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Thus the pathway does not rely on anti-bodies
having specific affinity towards a pathogen to
activate. Instead, it is constantly on the verge of
activating everywhere and is relying on self-
surface markers like fH to repress its activity.
This key regulatory role of fH in protecting self-
surfaces from attack by the innate immune
system makes it an attractive target for
microbial hijack. A strategy used by several
pathogenic bacteria such as Staphylococcus
aureus, Yersinia enterocolitica, Borrelia
burgdorferi and Streptococcus pneumoniae.8 In
2002 Ianelli et. al. identified 11 allelic variants
of PspC. They all sharing a similar domain
organization of an N-terminal fH binding
region, a helical region, a proline rich region
and a C-terminal anchor sequence. Ianelli et. al.
proposed to classify these variants into two
families A and B, distinct in the way they attach
to the bacterial outer membrane. The suggestion
was that Family A is defined by being anchored
with a C-terminal choline binding domain
whereas PspC in Family B is defined by being
anchored through a sortase dependent LPxTG
motif. 10 Since then, further study of PspC has
revealed that alleles of PspC can differ from
each other not only in the mechanism of
anchoring to the bacterial membrane but also in
the binding mechanism towards fH. In 2015 two
important articles were published illustrating
this fact. Achila et. al. were able to show that
PspC from the S. pneumoniae strain TIGR4
binds to the 9th CCP domain of fH through a
hydrophobic “lock and key” mechanism
mediated by the Tyr90 residue. 11 The same year,
Herbert et. al. showed that PspC from strain
D39 which lacks the Tyr90 residue binds the
same region of CCPs 8-10 as TIGR4 PspC,
despite sharing only 50% sequence similarity
and 6 out of 10 critical interface residues in the
binding site.12 This is a strong indication that
D39 PspC utilizes a different binding
mechanism than TIGR4 PspC, a mechanism
which is to current date unknown. Since the
both types of PspC, with and without Tyr90,
contribute to virulence it is of great interest to
elucidate the binding mechanisms of both.
Some strains of S. pneumoniae express more
than one allele of PspC where one such strain is
S. pneuminae_BHN418. Unpublished results by
Anuj Pathak of Birgitta Henrique Normark’s
Lab show that this strain expresses two PspC
alleles that are very similar to those expressed
by D39 and TIGR4 respectively. This property
makes it a good system for studying the
different functions of the two alleles and has
been the strain used for cloning PspC in this
project.
Figure 2 - PspC and its
interaction with Factor H.
A: General domain
organization of PspC and
protein sequence alignment
of [Tigr4]PspC and
[BHN418]PspC2 which
possess Tyr90 as contrasted
with [D39]PspC and
[BHN418]PspC1 which
lacks Tyr90. Sequence
alignments were performed
by the PROMALS3D web
server. 18 B: Factor H
structure model prediction
by the PHYRE2 server and
CCP domain organization.
C: N-terminal domain of
[TIGR4]PspC and its
interaction with fH CCP9
through a hydrophobic
“lock and key” mechanism
mediated by Tyr90.
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Anuj Pathak’s unpublished results reveal a
linear binding motif in [BHN418]PspC1
composed of residues K131-K139 and E149-
A154 in the N-terminal domain of
[BHN418]PspC1. Thus proving that the binding
site for fH is located on the N-terminal domain.
This master thesis report presents a project on
PspC1 isolated from S.pneumoniae strain
BHN418. From this point in this document
PspC1 refers to the PspC-allele of BHN418 that
is similar to [D39]PspC while PspC2 will refer
to the PspC-allele similar to [TIGR4]PspC in
their N terminal fH binding domain sequence.
The purpose of the project has been to elucidate
the mechanism of binding that provides PspC1
with affinity for fH. The method to accomplish
this has been to express recombinant PspC1 in
E. coli and to express individual fH CCP
domains as fusion proteins with Maltose
Binding Protein (MBP) in a prokaryote, E. coli
based expression system. Forcing expression of
eukaryote proteins in prokaryote systems is
often problematic, however this was the
approach used in the 2015 Achila. et. al study
which reports success in this endeavor.13
The long term potential of this research lies in
the possibility to increase understanding of
potential future protein vaccines against
S.pneumoniae. Purified PspC along with other
surface proteins like PspA has been shown to
have potential as antigens in protein vaccines
against pneumococcal infections. Even though
efforts to develop vaccines against
S.pneumoniae have been largely successful,
more than 90 distinct serotypes of S.
pneumoniae have been identified and current
vaccines are not effective against all of them.
Further efforts to understand and characterize
the bacterium is important in this effort.14
Materials and methods
Production of Recombinant Proteins All cloning was performed using the Sequence
and Ligation Independent Cloning (SLIC)
technique.15 In this technique genes are PCR-
amplified with primers designed to have
overhangs of around 20 bp that are homologous
to the intended vector sequence. All primer used
are indexed in Supplementary 1: Primers. The
vector construct was amplified as a linear
stretch of DNA. PCR-products were purified on
agarose gel with QIAquick PCR-cleanup kit by
Qiagen. Inserts and vectors were digested with
T4 DNA polymerase for 30 min to form 5’
sticky ends after which the digest was halted by
addition of a single dNTP. The vector and the
construct were incubated together in equimolar
amounts in 37°C for one hour before
transformation of the non-ligated intermediary
construct. Ligation and gap-filling occurs in in-
vivo and a completed vector was extracted with
QIAprep Spin Miniprep kit according to
protocol.
Transformation of the non-ligated intermediary
constructs was done with Mix’n’Go Competent
E.coli by Zymore Research, amplification of the
vector construct was done with XL-1 Blue
Figure 3 - SLIC-Procedure. Graphic adapted from 2007
M. Li and S. Elledge, Nature Methods.15
competent E.coli by Agilent. All plasmids were
controlled by sequencing before transformation
of the amplification and expression systems.
Protein production was performed in 1 liter
cultures of Terrific Broth supplemented with
100µg/ml ampicillin. Expression was induced
with 0.4mM Isopropyl β-D-1-
thiogalactopyranoside at OD600 = 0.4. Cell lysis
was performed by liquid homogenization. All
purified proteins were flash frozen in 10%
Glycerol and 20mM Hepes Buffer at pH 7.5 for
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storage and were thawed directly before use in
assays. Standard chemical were obtained from
Sigma Aldrich, AppliChem, VWR and
MERCK.
Recombinant PspC1
PspC1 was cloned from genomic DNA from S.
pneumoniae strain BHN_418. The gene was
cloned into a pET21d vector providing
ampicillin resistance, Lac-operon, a His6
purification tag and a TEV-cleaving site at the
N-terminal domain. The full length
PspC1_CBD construct was used for further
cloning to produce truncated fragments N1, N2
and N3. A chart detailing the recombinant
proteins can be seen in Figure 4.
Cell pellets were re-suspended in Lysis Buffer
B before cell disruption, buffer compositions
are detailed in Supplementary 2: Buffer
compositions. His-tagged recombinant PspC1
was purified on a 5 ml HisTrap FF column by
GE Healthcare, and eluted with 500mM
Imidazole according to protocol. Further
purification was performed using a 5 ml HiTrap
SP FF Ion Exchange Chromatography column
by GE Healthcare for proteins N1 and N2 due
to the theoretical pI of these constructs being 9.2
and 9.26 respectively. The longer N3 variant
was purified using size exclusion
chromatography on a HiLoad 16/600 Superdex
75 pg column by GE Healthcare since it had a
theoretical pI of 7.8.
fH CCP-MBP fusion proteins A vector construct of human fH synthesized by
Eurofins Genomics with optimized codons for
E.coli expression was used as the template for
cloning of the recombinant MBP-CCP fusion
constructs into a pET21d vector carrying an
ampicillin resistance module, Lac-operon,
Twin-Strep-tag purification tags by iba,
Maltose Binding Protein (MBP) and a TEV site
at the N-terminal domain. Four different CCP-
constructs were designed based on the results
from Achila et.al. 11 Each construct was made in
two versions, one with and one without a MalE
signal peptide for periplasmic expression at the
N-terminal domain. Cloning of additional CCP-
constructs comprising CCPS 1:7, 6:8, 8:10,
10:12, 13:15, 16:18 and 19:20 were also
prepared but were never transformed into an
expression system or used in any assays.
Small Scale Expression Test
Protein expression was tested in 2ml Terrific
Broth with 100µg/ml Ampicillin. Cultures were
incubated for 4 hours before induction with 0.4
mM IPTG at OD600= 0.7 and incubation over
night at 22°C. The entire culture volume was
pelleted and lysed with detergent based Lysis
Buffer B. The Protein was purified with
Figure 4 - Schematic view of produced PspC1 constructs. N signifies that the construct comprises the N-terminal domain
while CBD signifies Choline Binding Domain.
Figure 5 - Schematic overview detailing the CCP constructs. Eight constructs were produced, where four contained the
MalE signal peptide for periplasmic expression, hence the parenthesis around MalE in the schematic.
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MagStrep “type 3” XT beads by iba according
to protocol. SDS samples of the eluates were
run in parallel to compare the expression results
as seen in Figure 8. Expression was tested in
two strains of E.coli. SHuffle T7 Competent
E.coli (C3026) and SHuffle T7 Express
competent E.coli (C3029) by NEB. Both strains
constitutively express disulfide bond
reshuffling protein DsbC. At the point of testing
expression the CCP9 construct were not
completed since a completed CCP8:9 construct
was needed as a template. Hence expression
was tested for twelve cultures in total. Once the
expression strategy was chosen the CCP9
construct was cloned and expressed using the
same method. Large scale purification of the
fusion proteins was performed by resuspending
the cell pellet in Lysis Buffer A before cell
disruption followed by affinity chromatography
using a 5 ml Gravity flow Strep-Tactin XT
Superflow column by iba according to protocol.
Final purification was performed by Size
Exclusion Chromatography using a HiLoad
16/600 Superdex 75 pg column by GE
Healthcare.
Experiments
Pulldown assay
A preliminary assay of binding between CCP-
MBP fusion proteins and PspC1_N2 was
performed with a pulldown assay. Cell lysate
containing the recombinant MBP-CCP
constructs was incubated with MagStrep “type
3” XT beads by iba and washed according to
protocol but omitting the elution step resulting
in magnetic beads coated with CCP-MBP
fusion proteins. The bead slurry was divided
into two aliquots. One aliquot was eluted with
2.5mM Desthiobiotin in TBS and used as a
negative control while the other aliquot was
incubated with 1mg/ml His6-PspC1_N2 for one
hour before washing in TBS and elution. 1µl of
samples, negative control and positive control
(10ng/ml pure PspC1_N2) was blotted on
nitrocellulose membrane and allowed to air dry
for one hour before blocking over night with 1%
BSA. The membrane was washed with PBST
three times for 10 minutes each before
incubation with 1:4000 diluted HRP-coupled
Anti-6X His Tag antibody by abcam. An
identical blot was made for incubating with
1:4000 diluted HRP-coupled Anti-Strep-tag II
antibody by abcam. The membranes were
developed by coating the membranes in 750 µl
Pierce ECL Western Blotting Substrate by
Thermo Fisher according to protocol. Signal
was recorded with a BioRad ChemiDoc system.
Size Exclusion Chromatography Co-
purification
Recombinant MBP-CCP8 was cleaved by
overnight incubation in 4°C using TEV-
protease in Storage Buffer with addition of 1
mM EDTA, 3 mM Glutathione and 0.3 mM
Gluthathione disulfide to activate the TEV-
protease. The cleaved construct was purified
using on a Superdex 75 HR column by GE
Healthcare, and the fractions containing CCP8
were pooled and concentrated. CCP8 was
incubated with PspC1 constructs in equimolar
amounts and run over Superdex 75 HR column.
The resulting chromatogram was compared
with runs of PspC1 and CCP8 run separately at
the same respective concentrations. The assay
was performed with PspC1_N1 and PspC1_N2
constructs.
Figure 6 - Pulldown Assay. 1. Purification of CCP-MBP fusion proteins by affinity chromatography on MagStrep “type 3” XT
beads. 2. Addition of purified His6PspC1. 3. Elution from beads by addition of desthiobiotin. 4. Blotting on membrane and
signal detection by HRP-coupled antibodies.
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Figure 7 – Co-purification with Immobilized Metal Ion Chromatography and StrepTactin Affinity Chromatography. 1.
Recombinant PspC1_N3 was loaded onto the IMAC column and washed in 3x TBS. 2. Pure CCP-MBP fusion protein was
loaded onto the same IMAC column and washed in 3x TBS. The column was eluted with 500 mM imidazole. 3. The IMAC eluate
was loaded onto a Gravity flow Strep-Tactin XT Superflow column and washed in 3x TBS. 4. Strep-Tactin column was eluted
with addition of 2.5 mM desthiobiotin. Samples for analysis by SDS-PAGE were taken throughout experiment.
IMAC/StrepTag Co-purification
20µmol of His6-PspC1_N3 was loaded onto a
5ml Column Volume HisTrap FF by GE
Healthcare and washed with five column
volumes of TBS containing 25mM Imidazole
after which 15µmol of MBP-CCP8 in 800µl
HBS was loaded unto the same column and
washed with 5 CV. Elution was performed
using TBS containing 500mM Imidazole and
the eluate was collected. All fractions were
loaded onto a 5 ml Gravity flow Strep-Tactin
XT Superflow column by iba which was
washed in TBS 5x CV before eluting with
2.5mM Desthiobiotin in TBS. Flowthrough and
eluted fractions from the Strep-Tactin column
were pooled and concentrated 10 times before
analysis by SDS-PAGE. A schematic detailing
the procedure can be seen in Figure 7.
Ligand Tracer
The Ligand Tracer by Ridgeview Instruments is
a platform for measuring affinity interactions
between a receptor and a ligand. Measurements
take place on disposable petri-dishes onto which
the receptor is immobilized along with a
negative control and a blank at discrete
positions. The dish is placed at an angle in the
machine and the ligand is titrated into solution
at the bottom of the tilted dish. Measurements
take place at the top of the dish after it has been
rotated so the immobilized receptor passes
through the ligand solution. The ligand needs
fluorescent activity to be measured. In the ideal
case the fluorescent intensity measured at the
receptor position increases for every rotation
through the ligand-solution, and for every
increase in ligand concentration until the
receptor is saturated. At this point the ligand
solution is replaced with pure buffer and as the
measurements continue a dissociation curve can
be observed. 16
Samples of MBP-CCP8, MBP-CCP9 and full
length fH were spotted on petri dishes coated
with polydopamine and incubated overnight in
4°C. Care was taken not to let the spots flow
together. The spots were washed three times in
PBS and blocked with 1% BSA in PBS or
Pierce protein free blocking buffer by Thermo
Fisher, the dish was placed in the ligand tracer
instrument and filled with 3 ml PBS. Sequential
additions of fluorescently labeled PspC1_N3
was added with increasing concentrations at
approximately 100 minute intervals after which
the PBS solution containing PspC1_N3 was
replaced with pure PBS to measure any
dissociation curve. Binding activity was
monitored continuously throughout the
experiment at 488nm wavelength.
Microscale Thermophoresis
Microscale thermophoresis is a technique that
utilizes the thermophoresis phenomenon to
study particle interactions. Thermophoresis is
the phenomenon where particles migrate
directionally in small temperature gradients.
This migration depends on the size, charge and
hydration shell of the particle and is useful for
studying proteins since the thermophoretic
properties of a protein change as a function of
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conformational changes and affinity
interactions. The technique is made possible by
recent advances in optics and laser-physics
which allow very precise temperature gradients
to be induced by means of an infrared laser. The
thermophoretic movement is observed as
fluctuations of a fluorescent signal recorded at
the same point where the temperature gradient
is induced. This means that the particles
included in the experiment must have
fluorescent activity for a signal to be recorded.
In the case of making affinity interaction
measurements only one of the ligand or the
receptor needs to possess this activity. 17
Four measurements were taken; PspC1_N3
against fH twice, MBP-CCP8 against
PspC1_N3 and MBP-CCP9 against PspC1_N3.
The PspC1_N3 was titrated as a twelve point
1:2 dilution series starting at 125 nM and a
separate measurement was made with a three
point 1:2 dilution series in triplicate. Both
measurements were performed with a constant
concentration of 500 nM fH. MBP-CCP8 and
MBP-CCP9 was measured by titrating MBP-
CCPs in a twelve point 1:2 dilution series
starting at 500 nM, also with a constant
concentration of 500 nM fH. The experimental
set-up used the fluorescent activity of the CCPs
tryptophan residue.
Results
Expression and purification
MBP-CCP small scale expression test
Based on the expression test cytoplasmic
expression was chosen since it generally
produces better yields when compared to
periplasmic expression. Also, periplasmic
expression failed for CCP8 and CCP8:9 in the
C3026 strain. Theoretical molecular weights
correspond to results as analyzed by SDS-
PAGE. Degradation of fusion protein constructs
is evident by the presence of multiple bands.
MBP-CCP8
A population of proteins of similar molecular
weight are visible in both StrepTag AC and SEC
fractions. This indicates cleavage of the fusion
protein in vivo since the theoretical weight of
the CCP8 domain is 6.8kDa. The second lane in
the SEC-fractions after StrepTag Affinity
Chromatography gel is a sample from the void
volume. These are likely aggregated MBP-
CCP8 that have not been cleaved. The void
volume samples were also used in assays since
these were the only fractions containing pure,
un-cleaved MBP-CCP8.
Figure 8 - Expression test of
CCP constructs. The lanes
marked with yellow stars
correspond to the expression
strategy that was chosen.
SHuffle T7 Competent E.coli
(C3026) and SHuffle T7
Express competent E.coli
(C3029) by NEB were tested.
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Figure 9 - MBP-CCP8 purification results.
Figure 10 - MBP-CCP9 purification results.
Figure 11 –MBP-CCP8:9 purification results.
MBP-CCP9
The same population can be seen here as were
seen for CCP8. The first three lanes after the
ladder on the StrepTag AC elution fractions gel
are the complete lysate, the cleared lysate and
the flowthrough on the column. The first lane
after the ladder on the SEC fractions after Strep-
Tactin AC gel is the void volume. The same
tendency for aggregation can be seen for MBP-
CCP9 as for MBP-CCP8 although the band is
not as strong, indicating that the tendency for
aggregation is not as great.
MBP-CCP8:9
The same population is visible here as for MBP-
CCP8 and 9. The first two lanes after the ladder
on the StrepTag AC fractions gel are the lysate
and the column flow through.
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PspC1
Production of the truncated recombinant PspC1
was successful. The theoretical weight of
PspC1_N2 is 14.9 kDa and 42.1 kDa for
PspC1_N3. Analysis of produced proteins
match this within reason. A band corresponding
in weight to a dimer is present in purifications
of PspC1_N2. Purification of PspC1_N1
succeeded but no gels exist available for
presentation.
Figure 12 - PspC1 purification results. PspC1_N2: Both lanes after the ladder are samples of the pooled protein containing
fractions of the eluate from the respective columns. PspC1_N3: Lane 2 in the SEC-fractions after IMAC is the void volume.
Figure 13 - Pulldown
Assay Results
.
Pulldown Assay The results indicate binding between PspC1 and
CCP8 and not CCP9 as previously reported by
Achila et. al. 11 Binding to CCP10 could not be
ruled out since the Streptavidin positive control
gave a weak signal. The PspC1_N2 positive
control did not give any signal when developed
with Anti-His6 Ab, this might be due to the
concentration being very low. No signal was
observed from the CCP8:9 construct.
SEC Co-purification No significant decrease in the area of the peak
corresponding to CCP8 can be observed
coupled to an increase in area of any of the
PspC1 peaks for neither PspC1_N1, N2 nor N3.
No binding can thus be observed by analyzing
the overlayed chromatograms.
Figure 14 - SEC-Binding assay results.
PspC1_N2
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IMAC and StrepTag AC Copurification
Figure 15 –SDS-Page analysis of the copurification results.
Ligand Tracer
Figure 16 - Ligand Tracer results. On the left is the result from blocking with BSA and on the right is the result of protein free
block. Both plots show fluorescent signal with background subtracted.
Results indicate that PspC1_N3 has affinity for the full length Factor H. No strong conclusions can be
made due to poor signal to noise ratio, especially as PspC1_N3 concentration approaches 100nM. Before
this point, fH signal is consistently higher than both MBP-CCP8 and MBP-CCP9. Indications for
binding to CCP8 are also present since MBP-CCP8 is higher than MBP-CCP9. Protein free block
resulted in lower background and thus better signal to noise ratio, but still not good enough to make
draw strong conlusions.
PspC1_N3 does not show strong affinity for
MBP-CCP8 as is evident by the strong
signal from MBP-CCP8 in the flow through
of the IMAC as well as there being barely
any signal of any of the proteins in the
StrepTag AC eluate. Note that the eluate
and the flow through of the StrepTactin
column is concentrated 10x before loading
on the gel.
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Microscale Thermophoresis
Figure 17 –MST-results. Cap signifies the capillary number. Samples prepared as 12 point 1:2 dilution series and were loaded
from high to low concentration. Measurements with the MBP-CCPs start at 500nM PspC1_N3 and a constant concentration
of 250nM MBP-CCP. The measurements with fH are made with a higher constant concentration of 500nM fH. Upper left
illustrates PspC1_N3 and MBP-CCP8. MBP-CCP8. No affinity interactions can be observed, capillary 1 can be regarded as
an outlier. Upper right illustrates PspC1_N3 and full length fH. Clear signs of affinity interaction can be observed since
different concentration of PspC1_N3 give different signals. Lower left illustrates PspC1_N3 and MBP-CCP9. No affinity
interaction can be observed. Lower right illustrates the signal from PspC1_N3 by itself, capillary 1 can be considered an
outlier.
Affinity measurement calculations were
performed for the PspC1_N3 interaction with
full length fH resulting in a Kd of 248.6+70.4
nM, far from the single-digit nanomolar range
reported by Achila.et.al. 11
Discussion Expression of recombinant CCP constructs in
E.coli was a partial success. Peptides of the
correct weight were produced but it is unclear
whether the correct tertiary structure was
achieved by the prokaryote expression system.
Extensive degradation of the recombinant
MBP-CCP fusion construct was observed and
seems to have occurred in vivo. Analysis by
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SDS-PAGE as seen in Figure 9, Figure 10 and
Figure 11 are consistent with the degradation
being the result of hydrolysis of the TEV-site
since the CCPs have a theoretical molecular
weight of around 7 kDa. Redesigning the
constructs with a different protease site might
rectify this issue. Another good reason for using
a different protease site is that TEV-protease
requires a reducing environment to operate and
reducing environments could cause the
recombinant CCP to denature due to reducing
the cysteine bonds that stabilize its tertiary
structure.
The initial pulldown assay gave strong
indications of affinity interactions between
PspC1 and MBP-CCP8 but this result was not
reproducible by independent methods leading to
the conclusion that the pulldown assay result
was an artifact, despite being internally
reproducible. This is not entirely surprising
since neither the positive control of only PspC1
nor the MBP-CCP8:9 gave any signal. While
performing the experiments this was speculated
to be the result of steric hindrance of the binding
site, but that hypothesis does not explain the
lack of signal from the positive control.
However the positive control was heavily
diluted to a concentration of around 10ng/ml in
order to avoid its signal occluding the signal
from the adjacent spots. This concentration
might have been so low as to wash of and not
produce any signal whatsoever.
None of the PspC1 constructs showed affinity
towards CCP8 when co-purifying over SEC
with PspC1 or CCP runs at equimolar
concentration as reference, see Figure 16. The
first two experiments with PspC1_N1 and N2
were conducted with CCP8 as resulting from
treating MBP-CCP8 with TEV-protease. Strong
suspicions regarding the fold of the CCP8
domains were raised at this time and therefore a
third experiment was conducted with the
PSPC1_N3 construct and uncleaved MBP-
CCP8 since PspC1_N3 has a higher molecular
weight of 42.1 kDa as compared to PspC1_N2
at 14.9 kDa and PspC1_N1 at 12.8 kDa, the
MBP-CCP8 construct would not have to be
cleaved to resolve an affinity complex on the
column. If binding could be demonstrated under
these conditions one could draw the conclusion
that the TEV-cleavage denatured the CCP8
domain. No affinity interactions could be
observed however, leading to two possible
explanations. Either the CCP8 domain was
never properly expressed or PspC1 has affinity
for some other CCP-domain. In either case one
must conclude that the pulldown assay results
amount to an artifact. In retrospect the
constructs should have been redesigned after
performing the binding assays on the SEC-
column and not getting any significant
indications of binding. At the time the results
from the pulldown assay was considered so
convincing that work proceeded with the
original constructs
It seems likely that the lack of observable
affinity between the recombinant PspC1 and the
MBP-CCPs is due to the CCPs failing to
achieve proper tertiary structure since the
PspC1 is shown to have affinity for the full
length fH but not the CCPs. It seems likely that
this is due to a failure of the necessary disulfide
bonds to form. It is tempting to speculate that
this could also be an explanation for the
tendency for the CCPs to aggregate in the void
volume when doing SEC (see Figure 9) since
the molten-globule that likely results would
have exposed protein backbone and thus affinity
for copies of itself.
The production of PspC1 can be deemed a
success since binding was observed against full
length factor H during MST. PspC1 is a
prokaryote protein without complex tertiary
structure or cysteine bonds so this should also
not be problematic in theory. The truncation of
PspC1 could theoretically cause problems with
the tertiary structure but this was not observed.
The question of whether the shorter constructs
of PspC1, N1 and N2 are valid is still
unanswered since expression of these proteins
failed before the deadline for doing the MST-
measurements. Assessing whether these
constructs have affinity for full length fH would
be useful since shorter constructs are preferable
when producing co-crystals. An attempt to do
this with a pulldown assay was made by
utilizing magnetically coupled NHS-beads to
bind full length factor H. The assay was set up
according to protocol but the NHS-coupling
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Master Thesis Report Royal Institute of Technology Nils Lindström
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failed for unknown reasons. It was decided best
to abort the experiment and not spend time
optimizing since the protocol was rather time-
consuming.
The Ligand Tracer assays gave indications for
PspC1_N3 binding to full length fH. There was
also some indications for binding to MBP-
CCP8, but these suspicions were proven
unfounded by MST. It seems likely that
PspC1_N3 is very sticky since the background
noise was consistently higher than the signal.
This could also explain why it seemed like there
was affinity for MBP-CCP8. At higher
concentrations of PspC1_N3, approaching
100nM, the background noise became so great
as to completely occlude the signal, this also
made it impossible to calculate a wash-off curve
with any precision.
During Microscale Thermophoresis affinity
interactions were observed between PspC1_N3
and the full length fH but not between
PspC1_N3 and the two MBP-CCP constructs
that were tested, CCP8 and CCP9. All the runs
that included full length fH showed a strange
behavior in the beginning of the experiment
before the point of T-Jump where the
fluorescence signal was rapidly increasing. In
the typical case the signal stays constant before
T-Jump. One possible explanation for this
phenomenon that resembles photo bleaching in
reverse is that the fH aggregated at the capillary
walls rather than flowing freely in solution. All
experiments also produced noisy signals, which
might be due to the signal being dependent on
the fluorescent activity of tryptophan in the
CCPs and not a stronger fluorophore.
To proceed with the project from this point there
are several avenues to pursue. Trying to assay
binding with a Surface Plasmon Resonance
platform such as those provided by Biacore
seems reasonable since this type of
measurement might produce better data than
MST for this particular experimental set-up. It
might be a good idea to try the disulfide bond
reshuffling with DsbC in vitro as performed by
Achila et. al.11 on the produced constructs. The
T7 Shuffle Competent E.coli used as the
expression system constitutively expresses
DsbC, but results might differ if disulfide bond
reshuffling is performed ex-vivo. It also seems
reasonable to try the periplasmic expression
constructs since these are cloned and ready for
transformation, however the periplasmic
constructs also have the TEV cleavage site. If
neither of these approaches work it seems sound
to proceed by trying a eukaryote expression
system based on insect or leichmania cells.
While maintaining the cell culture is more
arduous as compared to a prokaryote cell
culture it might be a better option since it is
more likely to be able to express the
recombinant CCPs without an MBP-fusion.
This would reduce the work necessary to
produce the pure CCPs and also reduce the
metabolic strain on the expression system,
which in theory should increase the yield.
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Master Thesis Report Royal Institute of Technology Nils Lindström
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References 1. Anevlavis S, Bouros D. Community acquired bacterial pneumonia. Expert Opin Pharmacother.
2010;11(3):361-374. doi:10.1517/14656560903508770.
2. Pneumococcal Disease | Clinical | Streptococcus pneumoniae | CDC.
https://www.cdc.gov/pneumococcal/clinicians/streptococcus-pneumoniae.html. Accessed May
4, 2017.
3. Rodgers GL, Arguedas A, Cohen R, Dagan R. Global serotype distribution among Streptococcus
pneumoniae isolates causing otitis media in children: Potential implications for pneumococcal
conjugate vaccines. Vaccine. 2009;27(29):3802-3810. doi:10.1016/j.vaccine.2009.04.021.
4. French N, Gordon SB, Mwalukomo T, et al. A Trial of a 7-Valent Pneumococcal Conjugate
Vaccine in HIV-Infected Adults. N Engl J Med. 2010;362(9):812-822.
doi:10.1056/NEJMoa0903029.
5. O’Brien KL, Wolfson LJ, Watt JP, et al. Burden of disease caused by Streptococcus pneumoniae
in children younger than 5 years: global estimates. Lancet. 2009;374(9693):893-902.
doi:10.1016/S0140-6736(09)61204-6.
6. Schmidt CQ, Herbert AP, Kavanagh D, et al. A New Map of Glycosaminoglycan and C3b
Binding Sites on Factor H. J Immunol. 2008;181(4).
7. Dave S, Brooks-Walter A, Pangburn MK, McDaniel LS. PspC, a pneumococcal surface protein,
binds human factor H. Infect Immun. 2001;69(5):3435-3437. doi:10.1128/IAI.69.5.3435-
3437.2001.
8. Serruto D, Rappuoli R, Scarselli M, Gros P, Strijp JAG Van. Molecular mechanisms of
complement evasion: learning from staphylococci and meningococci. 2010.
http://dx.doi.org/10.1038/nrmicro2366.
9. Parham P. The Immune System.; 2014.
10. Iannelli F, Oggioni MR, Pozzi G. Allelic variation in the highly polymorphic locus pspC of
Streptococcus pneumoniae. Gene. 2002. doi:10.1016/S0378-1119(01)00896-4.
11. Achila D, Liu A, Banerjee R, et al. Structural determinants of host specificity of complement
Factor H recruitment by Streptococcus pneumoniae. Biochem J. 2015;465(2):325-335.
doi:10.1042/BJ20141069.
12. Herbert AP, Makou E, Chen ZA, et al. Complement Evasion Mediated by Enhancement of
Captured Factor H: Implications for Protection of Self-Surfaces from Complement. J Immunol.
2015;195(10). http://www.jimmunol.org/content/195/10/4986. Accessed May 17, 2017.
13. Achila D, Liu A, Banerjee R, et al. Structural determinants of host specificity of complement
Factor H recruitment by Streptococcus pneumoniae. Biochem J. 2015;465(2):325-335.
doi:10.1042/BJ20141069.
14. KALIN M. Pneumococcal serotypes and their clinical relevance. Thorax. 1998;53(3):159 LP-
162. http://thorax.bmj.com/content/53/3/159.abstract.
15. Li MZ, Elledge SJ. Harnessing homologous recombination in vitro to generate recombinant
DNA via SLIC. Nat Methods. 2007;4(3):251-256. doi:10.1038/nmeth1010.
16. Understanding how your ligand interacts with living cells.
http://www.ligandtracer.com/docs/LigandTracer Technology Note 1.6.pdf. Accessed August 31,
2017.
17. Jerabek-Willemsen M, André T, Wanner R, et al. MicroScale Thermophoresis: Interaction
analysis and beyond. J Mol Struct. 2014;1077:101-113. doi:10.1016/j.molstruc.2014.03.009.
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18. Pei J, Kim B-H, Grishin N V. PROMALS3D: a tool for multiple protein sequence and structure
alignments. Nucleic Acids Res. 2008;36(7):2295-2300. doi:10.1093/nar/gkn072.
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Master Thesis Report Royal Institute of Technology Nils Lindström
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Appendix
Supplementary 1: Primers Primers used for cloning are detailed here.
Melting temperatures were predicted by:
http://insilico.ehu.es/tm.php or
http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
Description Comment Overhang (homologous
recombination, 54°C, >
50°C?)
Iidentical to target Tm
of 60°C
Tmelt
(> 59°C)
Vec_R ACCCTGGAAGTAC
AGGTTTTCG
Vec_F TAACTCGAGGATC
CGGCTG
PspC1_F CCTGTACTTCCAGG
GT
GAAGAGGTTGGTG
GTAGGA
52.8
PspC1_F2 CCTGTACTTCCAGG
GT
GAAGAGGTTGGTG
GTAGGAATACC
56.8
PspC1N_REV1 404bp CCGGATCCTCGAGT
TA
GCGATCTTCTTCTT
TTTGATCCTCG
57
PspC1cbd_REV1 2075bp CCGGATCCTCGAGT
TA
GTTTACCCATTCAC
CATTGGCATTG
57.8
PspC1cbd_REV1 CCGGATCCTCGAGT
TA
GTTTACCCATTCAC
CATTGG
51.6
PspC1N2_REV1 1100bp CCGGATCCTCGAGT
TA
TTCTTCTGCTGCTT
TTCGTTTAGC
57
PspC_4k12_F CCTGTACTTCCAGG
GT
GGGCAAGATATAT
CGAAGAAGTATGC
56.1
PspC_4k12_R 290bp CCGGATCCTCGAGT
TA
TTCTGCAACTTTCT
TCTCAGCTTCTG
58.2
MBPcyto_F 1000bp GAAGAAGGTAAAC
TGGTAATCTGGAT
TAAC
56
MBPcyto_R AGTACAGGTTTTCT
GA
GTTATTGTTGTTGT
TGTTCGAGCTC
56
twSTII:vec_R 5600bp CCAGTTTACCTTCTT
C
GGAACCGCCACCG
GAC
58
http://insilico.ehu.es/tm.phphttp://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
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twSTII:vec_F TCAGAAAACCTGT
ACTTCCAGGG
56.5
twSTIIMBP:vec_
R
CATATGTTTCCTCC
TTTTGGATCTGATA
AATTG
twSTIIMBP:vec_
F
6760bp GGCTCTCGCCAAAA
TC
AGCGCGTGGAGCC
ATC
Peri_F 100bp AAGGAGGAAACATA
TG
AAAATAAAAACAG
GTGCACGCATCC
Peri_R GATTTTGGCGAGA
GCCGAGG
pET_MBP_R GCCCTGGAAGTAC
AGGTTTTC
55.7
pET_MBP_F TAACTCGAGGATC
CGGCTG
55.8
fHCCP1:7_F 1370bp CCTGTACTTCCAGG
GC
CGCCTGCTGGCTA
AAATCATTTG
58
fHCCP1:7_R CCGGATCCTCGAGT
TA
AGTTTTCACACGA
ATGCAGCG
57
fHCCP6:8_F 596bp CCTGTACTTCCAGG
GC
ACGCTGAAACCAT
GCGACTATC
58
fHCCP6:8_R CCGGATCCTCGAGT
TA
GCTTTTGATACAG
GTCGGTTGTG
57
fHCCP8:10_F 575bp CCTGTACTTCCAGG
GC
AAAACTTGTAGCA
AAAGCTCTATTGA
CATC
57
fHCCP8:10_R CCGGATCCTCGAGT
TA
TTGTTCTTTACAGA
TCGGCAAATCAG
57
fHCCP10:12_F 398bp CCTGTACTTCCAGG
GC
CAAAGTTGTGGCC
CTCCG
56
fHCCP10:12_R CCGGATCCTCGAGT
TA
CTTATCGATGGCA
ACGCATTG
55.4
fHCCP13:15_F 566bp CCTGTACTTCCAGG
GC
AAGAAATGCAAAT
CTTCTAACCTTATC
ATCC
56
fHCCP13:15_R CCGGATCCTCGAGT
TA
ACCTTCGCACTGT
GGCG
58
fHCCP16:18_F 569bp CCTGTACTTCCAGG
GC
GAAGGTTTGCCCT
GCAAATC
55
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fHCCP16:18_R CCGGATCCTCGAGT
TA
GGAGTCTTTGCAT
TGCGGC
57.5
fHCCP19:20_F 413bp CCTGTACTTCCAGG
GC
TCCACAGGCAAAT
GCGG
55
fHCCP19:20_R CCGGATCCTCGAGT
TA
CCGTTTCGCACAA
GTCGG
57
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Supplementary 2: Buffer compositions
Lysis Buffer A
Gycerol 10% by volume
pH 7.5 Hepes 50 mM
NaCl 500 mM
MgCl2 1 mM
DNAse 40 µg/ml
Lysozyme 500 µg/ml
PMSF 1 mM
1 tablet cOmplete Ultra
protease inhibitor.
In 50ml total volume
dH20 To 50ml
Lysis Buffer B
B-PER II Bacterial Protein
Extraction Reagent by
Thermo Fisher.
To 50ml
DNAse 40 µg/ml
Lysozyme 500 µg/ml
PMSF 1 mM
1 tablet cOmplete Ultra
protease inhibitor.
In 50ml total volume
Storage Buffer
Glycerol 10% by volume
pH 7.5 Hepes 20 mM
NaCl 300 mM
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Supplementary 2: Protein sequences of produced constructs.
Orange – StrepTag or His6-Tag, Grey – Peptide linker, Blue – Maltose Binding Protein, Red – TEV-
site, Black – GOI.
twSTII-MBP-TEV-CCP8
SAWSHPQFEKGGGSGGGSGSAWSHPQFEKSGGGSEEGKLVIWINGDKGYNGLAEVGKKFEK
DTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF
TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA
FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKE
FLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGKTCSKSSIDIENGFISESQ
YTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKS
twSTII-MBP-TEV-CCP8:9
SAWSHPQFEKGGGSGGGSGSAWSHPQFEKSGGGSEEGKLVIWINGDKGYNGLAEVGKKFEK
DTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF
TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA
FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKE
FLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGKTCSKSSIDIENGFISESQ
YTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSCDIPVFMNARTKNDFTWF
KLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERE
twSTII-MBP-TEV-CCP9
SAWSHPQFEKGGGSGGGSGSAWSHPQFEKSGGGSEEGKLVIWINGDKGYNGLAEVGKKFEK
DTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF
TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA
FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKE
FLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGSCDIPVFMNARTKNDFT
WFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERE
twSTII-MBP-TEV-CCP10
SAWSHPQFEKGGGSGGGSGSAWSHPQFEKSGGGSEEGKLVIWINGDKGYNGLAEVGKKFEK
DTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF
TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA
FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKE
FLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
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YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGERECELPKIDVHLVPDRK
KDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPICKEQ
His6-TEV-PspC1_N1
HHHHHHENLYFQGGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTVALINELQDIKKTYL
YNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAE
His6-TEV-PspC1_N2
HHHHHHENLYFQGEEVGGRNTPTVTSSGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTV
ALINELQDIKKTYLYNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAEA
KKKAEDQKEEDR
His6-TEV-PspC1_N3
HHHHHHENLYFQGEEVGGRNTPTVTSSGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTV
ALINELQDIKKTYLYNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAEA
KKKAEDQKEEDRRNYPTNTYKTLELEIAESDVKVKEAELELVNEEAKPGNEEKIKKAKAKVE
SEKAEAIRLEEIKTDREEAKRKADAKLKEAVENNAATSEQGEPKRRVKRGVLGEPATPDKKE
NDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKAKDQKEEDRRNYPTNTYKTLELEIAESDVKV
KEAELELVKEEAKESRNEEKVKQAKAKVESKKAEATRLEKIKTDRKKAEEAKRKAAEE
His6-TEV-PspC1_CBD
HHHHHHENLYFQGEEVGGRNTPTVTSSGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTV
ALINELQDIKKTYLYNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAEA
KKKAEDQKEEDRRNYPTNTYKTLELEIAESDVKVKEAELELVNEEAKPGNEEKIKKAKAKVE
SEKAEAIRLEEIKTDREEAKRKADAKLKEAVENNAATSEQGEPKRRVKRGVLGEPATPDKKE
NDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKAKDQKEEDRRNYPTNTYKTLELEIAESDVKV
KEAELELVKEEAKESRNEEKVKQAKAKVESKKAEATRLEKIKTDRKKAEEAKRKAAEEDKV
KEKPAEQPQPAPAPQPEKPAPKPEKPAPAPKPENPAEQPKAEKPADQQAEEDYARRSEEEYNR
LTQQQPPKTEKPAQPSTPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNSNGAMA
TGWLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANG
SMATGWLQNNGSWYYLNANGSMATGWLQYNGSWYYLNANGSMATGWLQYNGSWYYLN
SNGAMVTGWLQNNGSWYYLNANGSMATDWVKDGDTWYYLEASGAMKASQWFKVSDKW
YYVNGSGALAVNTTVDSYRVNANGEWVN
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