Prokaryotic expression of human complement regulator ...1147592/FULLTEXT01.pdf · Master Thesis...

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

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.

Master Thesis Report Royal Institute of Technology Nils Lindström

1

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


2

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.


3

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


4

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.


5

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.


6

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


7

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.


8

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.


9

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


10

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.


11

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


12

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


13

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.


14

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.


15

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alignments. Nucleic Acids Res. 2008;36(7):2295-2300. doi:10.1093/nar/gkn072.


16

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/


17

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


GC

ACGCTGAAACCAT

GCGACTATC

58


TA

GCTTTTGATACAG

GTCGGTTGTG

57


GC

AAAACTTGTAGCA

AAAGCTCTATTGA

CATC

57


TA

TTGTTCTTTACAGA

TCGGCAAATCAG

57


GC

CAAAGTTGTGGCC

CTCCG

56


TA

CTTATCGATGGCA

ACGCATTG

55.4


GC

AAGAAATGCAAAT

CTTCTAACCTTATC

ATCC

56


TA

ACCTTCGCACTGT

GGCG

58


GC

GAAGGTTTGCCCT

GCAAATC

55


18


TA

GGAGTCTTTGCAT

TGCGGC

57.5


GC

TCCACAGGCAAAT

GCGG

55


TA

CCGTTTCGCACAA

GTCGG

57


19

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


20

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







YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGKTCSKSSIDIENGFISESQ

YTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTCIKSCDIPVFMNARTKNDFTWF

KLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERE

twSTII-MBP-TEV-CCP9







YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGSCDIPVFMNARTKNDFT

WFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPICYERE

twSTII-MBP-TEV-CCP10








21

YAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNSENLYFQGERECELPKIDVHLVPDRK

KDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPICKEQ

His6-TEV-PspC1_N1

HHHHHHENLYFQGGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTVALINELQDIKKTYL

YNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAE

His6-TEV-PspC1_N2

HHHHHHENLYFQGEEVGGRNTPTVTSSGQDISKKYADEVESHLKKILSEIQTQLDRKRHTKTV

ALINELQDIKKTYLYNLNVLKEKSELPSKIKAKLEVAFDQFKKDTLKPGEKVAEAEKKVAEA

KKKAEDQKEEDR

His6-TEV-PspC1_N3



KKKAEDQKEEDRRNYPTNTYKTLELEIAESDVKVKEAELELVNEEAKPGNEEKIKKAKAKVE

SEKAEAIRLEEIKTDREEAKRKADAKLKEAVENNAATSEQGEPKRRVKRGVLGEPATPDKKE

NDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKAKDQKEEDRRNYPTNTYKTLELEIAESDVKV

KEAELELVKEEAKESRNEEKVKQAKAKVESKKAEATRLEKIKTDRKKAEEAKRKAAEE

His6-TEV-PspC1_CBD



KKKAEDQKEEDRRNYPTNTYKTLELEIAESDVKVKEAELELVNEEAKPGNEEKIKKAKAKVE

SEKAEAIRLEEIKTDREEAKRKADAKLKEAVENNAATSEQGEPKRRVKRGVLGEPATPDKKE

NDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKAKDQKEEDRRNYPTNTYKTLELEIAESDVKV

KEAELELVKEEAKESRNEEKVKQAKAKVESKKAEATRLEKIKTDRKKAEEAKRKAAEEDKV

KEKPAEQPQPAPAPQPEKPAPKPEKPAPAPKPENPAEQPKAEKPADQQAEEDYARRSEEEYNR

LTQQQPPKTEKPAQPSTPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNSNGAMA

TGWLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANG

SMATGWLQNNGSWYYLNANGSMATGWLQYNGSWYYLNANGSMATGWLQYNGSWYYLN

SNGAMVTGWLQNNGSWYYLNANGSMATDWVKDGDTWYYLEASGAMKASQWFKVSDKW

YYVNGSGALAVNTTVDSYRVNANGEWVN

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