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Characterization of IL-1β secretion by fusing elastin-like polypeptides to pro-caspase-1

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Characterization of IL-1β secretion by fusing elastin-like polypeptides to pro-caspase-1

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CHARACTERIZATION OF IL-1β SECRETION BY FUSING
ELASTIN-LIKE POLYPEPTIDES TO PRO-CASPASE-1

by

Yutong Wang

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2020

Copyright 2020 Yutong Wang
ii
ACKNOWLEDGEMENTS
I would first like to express my gratitude to my supervisor Dr. Curtis Okamoto for accepting me
to study in his lab and giving me the chance to have my own research project. Thanks to the trust
he gave me during the past two years, so that I have many opportunities to think independently
and improve my experimental skills. At the same time, he also gave me many useful instructions
about my own project, graduate courses and also future study plans. Without his help, my
graduate study would be much more difficult than it is now.

I extend my gratitude towards my committee members Dr. Andrew MacKay and Dr. Ian
Haworth for being a part of my thesis committee. They have provided useful suggestions on my
project and also feedback to help complete my thesis. At the same time, thanks to Dr. MacKay’s
lab for providing some necessary reagents and equipment during my project.

In addition, I am grateful to Anh Truong, Taojian Tu and Yue Zhang, the Ph.D. students in Dr.
Okamoto’s lab. Anh taught me how to think and design the project scientifically and logically
and helped me construct the basic framework of my project. Taojian taught me some basic lab
skills and also the cell culture with great patience. Yue Zhang helped me a lot while ordering
reagents. I would also like to thank Vishvesha Vaidya, another Masters student in the same lab,
for discussing issues with me when I was facing difficulties.

Finally, I must thank my parents. Their support and encouragement are important driving forces
for me to successfully complete the Masters Program. Without them, I would not have the
iii
chance to study in USC and would not have enough confidence to continue pursuing Ph.D. study.
Also thank you to my friends who have provided me support and help during the past two years.

iv
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ......................................................................................................ii
LIST OF FIGURES .................................................................................................................. v
ABSTRACT ............................................................................................................................. vi
CHAPTER ONE: INTRODUCTION ...................................................................................... 1
1.1 Interleukin-1β .................................................................................................................... 1
1.2 Inflammasomes ................................................................................................................. 2
1.3 Secretion of IL-1β ............................................................................................................. 7
1.4 Caspase-1 .......................................................................................................................... 8
1.5 Elastin like polypeptides (ELP) fused Caspase-1 ............................................................. 10
CHAPTER TWO: MATERIALS AND METHODS ............................................................. 12
2.1 Materials ......................................................................................................................... 12
2.2 Solutions and Reagents.................................................................................................... 13
2.3 Methods .......................................................................................................................... 15
2.3.1 Create ELP-CASP1 Fused Plasmids by Cloning Methods ......................................... 15
2.3.2 ELP-CASP1 expression ............................................................................................ 17
CHAPTER THREE: RESULTS ............................................................................................ 21
3.1 Creation of ELP-CASP1 Plasmids ................................................................................... 21
3.2 Protein Expression .......................................................................................................... 26
CHAPTER FOUR: DISCUSSION ......................................................................................... 33
REFERENCES ....................................................................................................................... 38

v
LIST OF FIGURES
Figure 1. Two-signal model for inflammasome activation 4
Figure 2. Homotypic domain interactions direct NLRP3 inflammasome assembly 6
Figure 3. A. Domain structure of Caspase-1 B. Potential dimeric species of Caspase-1
43
. 9
Figure 4. Structure of ELP ligated pro-Caspase-1 11
Figure 5. Size of EcoRI @HF Digested pCl-caspase1 Plasmids. 21
Figure 6. Positive E.coli colonies of oligo-CASP1 plasmids. 22
Figure 7. AfeI verification of oligonucleotide insertion 23
Figure 8. AcuI and NdeI digested ELPs plasmids. 24
Figure 9. Positive E.coli colonies of ELP-CASP1 plasmids . 25
Figure 10. HinDIII @HF restriction digest to verify ELP insert ligation. 25
Figure 11. Western blot to determine the expression of ELP-CASP1 fusion protein in THP-1
cells grown in 1% FBS RPMI 1640 medium. 27
Figure 12. Western blot to determine the expression of ELP-CASP1 fusion protein in HEK293
cells grown in 1% FBS DMEM medium. 28
Figure 13. Western blot to determine the expression of ELP-Casp1 fusion proteins in HEK293T
cells grown in 10% FBS RPMI 1640 medium. 29
Figure 14. Western blot to determine the expression of ELP-Casp1 fusion proteins in HEK293T
cultured in 1% FBS RPMI 1640 medium. 31

vi
ABSTRACT
Innate immunity is an important part of the immune system which can be triggered by pathogen-
associated molecular patterns and can cause the production of proinflammatory cytokines and
chemokines to protect the human body. Interleukin 1β (IL-1β) is the best-characterized member
of the interleukin-1 cytokine family. It has many biological effects including T-cell activation, B-
cell activation, homeostasis regulation and promotion of some immune diseases. IL-1β is
expressed as 31 kDa proprotein and can be activated through the process of Caspase-1
proteolytic activation based on the inflammasomes platform. Triggered by different pathogens,
intracellular inflammasomes are assembled with three parts including the sensor molecule, the
enzymatic proteolytic component (pro-Caspase-1) and the adaptor molecule (ASC). After being
recruited to inflammasomes, pro-Caspase-1 can form homodimers and be activated through
autoproteolysis. The active Caspase-1 then cleaves proIL-1β to its active form. However, the
secretion mechanism of active IL-1β is still not clear. Elastin like polypeptides (ELPs) are
repetitive polypeptides that are derived from human tropoelastin and exhibit temperature-
dependent phase transition. To investigate the secretion of IL-1β, different ELPs were designed
to be fused to pro-Caspase-1 to control IL-1β activation and secretion through changing culture
temperature in the absence of other parts of inflammasomes. The protein ligation was carried out
based on the cloning method (plasmid construction). The plasmids encoding ELP-CASP1
(elastin like polypeptides and Caspase-1) fusion proteins were transiently transfected into both
THP-1 and HEK293 cell lines and were analyzed for their expression by western blot. After
trying several times, Caspase-1 was successfully expressed in HEK293T cells. And the 100 kDa
band which corresponds to the molecular weight of A96-CASP1 was detected. This band might
vii
represent the expressed A96-CASP1 which need to be confirmed in the future. If the fusion
protein is successfully expressed, it may be possible to artificially control cytokine activation and
secretion by this approach.
1
CHAPTER ONE: INTRODUCTION
1.1 Interleukin-1β
Nonspecific innate immunity and specific adaptive immunity together form the mammalian
immune system that protects humans from pathogens and danger signals. In the innate immune
system, pathogen-associated molecular patterns (PAMPs) are recognized by the germline-
encoded pattern-recognition receptors (PRRs) which are expressed constitutively with a limited
number in the host. After that, a series of signals will be sent to trigger the production of
proinflammatory cytokines and chemokines which can attract or support invading macrophages
or neutrophils.
1

Interleukin-1 (IL-1) is a cytokine family that plays important roles in innate immunity and can
affect nearly all cell types. The study of IL-1 was started in 1943 to investigate leukocyte-
produced endogenous proteins that led to fever. 40 years later, in 1984, by cDNA cloning, two
distinct cytokines IL-1α and IL-1β were first identified. Now there are already 11 members
characterized in the IL-1 family.
54

Among this family, interleukin-1β (IL-1β) is the best characterized one. IL-1β is encoded by the
IL-1β gene and expressed as a 31 kDa proprotein (Pro-IL-1β ) in activated macrophages. Pro-IL-
1β is inactive and remains in the cytosol until the cleavage of the aspartic-alanine peptide bond
by Caspase-1 which was also previously named as the IL-1β converting enzyme (ICE).
2
IL-1β
has a β-trefoil fold composed of 12 anti-parallel β-strands which is conserved among other IL-1
family cytokines
55
. It binds to the IL-1receptor (IL-1R) and activates downstream signals for
different inflammatory responses as well as for some autoimmune diseases.
2

IL-1β has a wide range of biological effects. For immunological aspects, it participates in the
process of T-cell activation, IL-2R expression, B-cell activation via induction of IL-6 and also
lymphokine gene expression. For its pro-inflammatory properties, IL-1β plays important roles in
homeostasis by regulating temperature, sleep, and feeding
3
, as well as other processes such as
neuropeptide release, endothelial cell activation, hypotension, and beta islet cell cytotoxicity
4
.

Except for normal homeostatic functions, the overproduction of IL-1β is also involved in many
different disease states. Clinical trials and in vitro experiments provided evidence that higher
levels of IL-1β have been detected in patients with sepsis, rheumatoid arthritis, atherosclerosis,
and asthma, compared to healthy volunteers.
5
In addition, IL-1β also exhibits tumor-promoting
effects. Overproduction of IL-1β promotes carcinogenesis and metastasis by inducing
angiogenesis, endothelial cell activation, and suppression of adaptive antitumor immunity
through promoting the activation of myeloid-derived suppressor cells (MDSCs) and sustaining
the immunosuppressive activity of tumor-associated macrophages (TAM)
6,7
. Many studies in
recent years indicate that the activation and secretion of IL-1β is closely related to the activation
of inflammasomes, especially of the NLRP3 inflammasome.

1.2 Inflammasomes
Inflammasomes are multimeric protein complexes acting as essential components of innate
immunity. Activation of inflammasomes can help clear pathogens and damaged cells by
triggering maturation and secretion of IL-1β and inducing pyroptosis. Pyroptosis is a form of
programmed cell death that occurs upon infection triggered by intracellular pathogens and is
3
mediated by the enzyme Caspase-1. Inflammasomes are composed of three parts: the sensor
molecule, the enzymatic component (Caspase-1), and the adaptor molecule (ASC)
12
.

Based upon their structures, the inflammasome sensors are divided into three groups: the
nucleotide-binding domain-like receptors (NLRs) family, the absent in melanoma 2 like
receptors (ALR) family, and the Pyrin family. NLR family members, from their N-terminal to C-
terminal, all contain a variable domain, a nucleotide-binding domain (NBD) and a leucine-rich
repeat (LRR)
12
. NLRP3 is the best characterized one of the NLR family. The absent in
melanoma 2 (AIM2) inflammasome is an important member of the ALR family. It has a HIN200
(HIN is the abbreviation of hematopoietic expression, interferon-inducible nature, and nuclear
localization) domain directly binding to cytosolic double-stranded DNA (dsDNA) and a pyrin
domain (PYD) that distinguishes it from other ALRs by its ability to interact with ASC and form
an inflammasome
13,14
. And there are also the recently identified Pyrin inflammasomes. Pyrin is
encoded by the mutated gene in patients with familial Mediterranean fever (FMF) and can detect
the change in activity of the small ras-like GTPase RhoA
15
.

The maturation of IL-1β is a complicated process under tight regulation. The process is usually
explained by using the NLRP3 inflammasome as a model, as it is the best-characterized
inflammasome. The entire process can be divided into the priming step and the activation step,
with each step induced by distinct signals
9
(Fig1).
4

Figure 1. Two-signal model for inflammasome activation
11
.
Signal 1 (left) is provided by microbial molecules and endogenous cytokines which leads to the activation of the transcription
factor NF-kB and upregulation of NLRP3 and pro-IL-1β. Signal 2 (right) is provided by a plethora of stimuli, including ATP,
pore-forming toxins, particulate matter and viral RNA. The activation of inflammasomes results in multiple effects, such as K
+

efflux, mitochondrial dysfunction, cytosolic ROS and lysosome leakage.
11

Signal 1 (Fig. 1) stimulates the transcription and translation of the IL-1β gene and other
components of inflammasomes (priming step). When cells, such as macrophages, are treated
with Toll-like receptor (TLR) agonists including any of the microbial components,
lipopolysaccharide (LPS), CpG dinucleotides, lipoproteins, or the drug phorbol 12-myristate 13-
acetate (PMA), TLRs, especially TLR4 and TLR2, will generate signals to activate the
transcription factor NF-kB. A set of Toll-Interleukin Receptor (TIR) domain-containing adaptors
such as myeloid differentiation primary response gene 88 (MyD88) and MyD88-adaptor-like
(TIRAP/MAL) are recruited by TLRs. Those adaptors can trigger the phosphorylation and
degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IkB)
5
proteins which is a necessary step in the activation of NF-kB.
8
Activated NF-kB then enhances
the expression of both pro-IL-1β and other NLRP3 inflammasome components. Furthermore,
gene transcription of pro-IL-1β can also be triggered by inflammatory cytokines such as tumor
necrosis factor (TNF) and IL-1β itself through the TNF receptor (TNFR) and IL-1R pathways
9,10
.

The second signal triggers the assembly of the NLRP3 inflammasome and maturation of IL-1β.
Instead of physically interacting with activators, NLRP3 is activated by sensing cellular signals
induced by NLRP3 activators. The second signal is provided by a rise in intracellular ATP, viral
RNA, pore-forming toxins, and particulate matter sensed by different receptors, such as P2X7R
11
,

on the cell membrane. In response to NLRP3 stimuli, K
+
efflux is sufficient to activate the
NLRP3 inflammasome by lowering the intracellular K
+
concentration
16
. Ca
2+
mobilization is
crucial in NLRP3 activation triggered by the signal of ATP-stimulated mitochondrial damage
17
.
Mitochondrial dysfunction and cytosolic ROS (Reactive Oxygen species) produced by NADPH
(the reduced form of nicotinamide adenine dinucleotide phosphate, abbreviated NADP+) oxidase
are believed to be implicated in the activation of NLRP3 inflammasome at first based on studies
showed that chemical inhibitors of the voltage-dependent anion channel (VDAC) can suppress
the activation of inflammsomes. This was because the uptake of Ca
2+
into mitochondria is
inhibited as well as the mitochondria metabolic activity.
18,19
Cathepsin B is a lysosomal cysteine
protease. It is released into the cytosol when the lysosome membrane is damaged, which may be
caused by presence of particulate matter. Cathepsin B also gives rise to NLRP3 activation based
on researches that showing CA-074-Me, the chemical inhibitor of cathepsin B, can inhibit
NLRP3 inflammasome activation
20,21
However, the exact mechanisms of mitochondria
dysfunction, cytosolic ROS and lysosome leakage in NLRP3 activation are still unclear.
6

Figure 2. Homotypic domain interactions direct NLR P3 inflammasome assembly
22
.
NLRP3 inflammasomes are assembled through two steps of homotypic domain interactions. The sensor molecule and the adaptor
protein ASC are attached together through PYD-PYD interaction. Then ASC recruits pro-Caspase1 through CARD-CARD
interaction

After the second signal stimulation, the NLRP3 inflammasome is assembled through two steps of
nucleation-polymerization through homotypic protein domain interactions
23
(Fig. 2). The sensor
molecule NLRP3 is a tripartite protein containing an N-terminal Pyrin (PYD) domain, a
nucleotide-binding NTPase domain (NACHT) which is named by abbreviation of proteins that
contain it and an LRR domain
24
. The NACHT domain is thought to have ATPase activity, and
mutations in this domain reduce ATP binding and Caspase-1 actiavtion.
25
The adaptor protein
ASC contains a PYD domain and a caspase activation and recruitment domain (CARD). The
enzymatic pro-Caspase-1 component also contains a CARD domain. For the first step, NLRP3s
are oligomerized into a ring-like platform based on the interaction among its NACHT domains
26
.
For this ring-like structure, the PYD domains are in the center and the LRR domains are pointing
to the outside. Then ASC is recruited through the PYD-PYD interaction to form the NLRP3
nucleated core. Second, ASC could amplify the signal by recruiting cytosolic pro-Caspase-1
through CARD-CARD interactions. The formation of a pro-Caspase-1 filament (Fig. 2)
significantly increases its local concentration resulting in Caspase-1 dimerization and
activation
23,27
.
7

1.3 Secretion of IL-1β
Between the 1940s and 1950s, the mechanism of protein secretion, named later as the
conventional protein secretion pathway, was first elucidated and showed that most proteins are
secreted through the endoplasmic reticulum (ER) and Golgi system
28
. The first step of the co-
translational secretory pathway is to detect the N-terminal signal sequence of the nascent peptide
through the signal recognition particle (SRP). The nascent peptide chain binds to the SRP
receptor on the ER membrane resulting in the translocation of the nascent protein chain into the
ER lumen through the protein conduction channel
28-30
of the SRP receptor. Several things happen
in the ER in sequence, such as the signal sequence is cleaved, the protein is folded by chaperones,
and the folded protein binds to the coat protein complex II (COPII). The COPII coated vesicle
then moves to the Golgi apparatus and releases the protein to arrive ultimately different
destinations.
28

However, by sequencing cDNA of the interleukin-1 precursor, IL-1β was found to have no signal
sequence on its N-terminus, thus suggesting that it is secreted through an unconventional
secretion pathway. This suggestion was supported by the insensitivity of IL-1β secretion to
conventional secretion pathway blocking drugs
31,32
, such as Brefeldin A. Although the IL-1β
secretion mechanism is still unknown, some hypotheses have been proposed to explain its
apparent unconventional secretion. In an early model, a portion of pro-IL-1β and pro-Caspase-1
are translocated into secretory lysosomes, and the activated proteins are secreted through
lysosomal exocytosis
32
. The second model indicated that the cytokine is secreted upon lysis of
the stimulated cell membrane, resulting in the shedding of microvesicles
33
. In 2007, the exosome
model was proposed: multivesicular bodies release the cytokine based on exosomal secretion
34
.
8
Common among the three models is that activation of Caspase-1 enzyme and cytokine both
happen within intracellular vesicular structures
35
. However, those models still remain
controversial. Later the autophagy secretion model suggested that autophagosomes can capture
pro-IL-1β for autophagic degradation and control the release of active cytokine, which is
triggered by TLR agonists
36
. In recent years, real-time single-cell imaging provided evidence for
the hypothesis that secretion of cytokine IL-1β is related to cell death such as pyroptosis caused
by Caspase-1
37
.

A gasdermin D (GSDMD)-dependent unconventional secretion model was also developed
recently. Caspase-1 specifically cleaves the linker between GSDMD-N domain and GSDMD-C
domain, which liberates the N-terminal domain from intramolecular inhibition and results in cell
pyroptosis
38
. The free GSDMD-N domains insert into the lipid bilayer of the plasma membrane
and form gasdermin pores with 10-14 nm inner diameter by recognizing and binding to PI(4,5)P2
in the inner leaflet
39
. Because of the formation of gasdermin pores in the plasma membrane, cells
lose their ionic homeostasis quickly which results in pyroptosis, and in the meanwhile, 4.5 nm
diameter IL-1β can also be secreted
40
.

However, all models mentioned here have been only hypothesized, and more research is needed
to confirm any of them.

1.4 Caspase-1
In 1989, a previously undescribed catalytic reaction that cleaved proteins and peptides before
aspartic acid residues was identified to be catalyzed by a single protease which showed much
9
higher cleavage activity on proIL-1β compared to other proteases
41
. This protease was named as
Caspase-1 later. In 1992, the primary structure, the catalytic mechanism, and role in the
maturation of pro-IL-1β of Caspase-1 were defined for the first time
42
. It is a 45 kDa cysteine
protease composed of a large subunit (p20) and a small subunit (p10), linked by an interdomain
linker (IDL), and a death fold CARD domain on the N-terminus, separated by a CARD domain
linker (CDL)
42,43
(Fig. 3A).

Although only the p20 subunit has catalytic activity, both p20 and p10 are required to form an
active Caspase-1 heterodimer complex when processing pro-IL-1β. Recruitment by the ASC hub
through a CARD-CARD interaction (Fig. 2) enables the dimerization of Caspase-1 based on
small subunit-small subunit (SS-SS) interaction (p46 species, Fig. 3B) through an increase in
local protein concentration
44
. Dimerized Caspase-1 undergoes auto-proteolysis at different Asp-
Xaa bonds within the IDL or CDL (shown in Fig. 3A), and the cleavage in IDL that generates the
p35 and p10 subunits gives rise to the cytokine processing activity
45
. Activated Caspase-1
processes the pro-IL-1β via two steps, a 27 kDa inactive intermediate is generated first by the
cleavage at Asp 27 (D27 in single letter amino acid code), and then it gives rise to the bioactive
17 kDa IL-1β by cleavage at D116
46
.

Figure 3. A. Domain structure of Caspase-1 B. Potential dimeric species of Caspase-1
43
.
(A) Structure of pro-Caspase1 and possible self-processing sites within the CDL and IDL. (B) Potential species of dimeric
Caspase-1 generated by IDL or CDL cleavage.43
10
Although the recombinant tetramer of p20/p10 as the catalytically active form was identified
long ago, the active species in cells was not defined until 2018
47
. The transient p33/p10 species
(Fig. 3B) is the predominantly active form of Caspase-1. Then the p20/p10 species is generated
from the active form by self-cleavage. This is the deactivation step with different kinetics in
different cell types or dependent upon the differing size of inflammasomes.
47

1.5 Elastin like polypeptides (ELP) fused to Caspase-1
Elastin like polypeptides (ELPs) are derived from human tropoelastin. Tropoelastin is the 72 kDa
soluble precursor molecule of natural elastin. Elastin is the extracellular matrix protein found in,
and provides elasticity to, arteries, lungs, and skin. Tropoelastin contains two different domains,
the hydrophobic domain formed by the repeated amino acids sequence of Gly, Val, Ala and Pro,
and the hydrophilic domain is mainly composed of Lys and Ala. The covalent interchain
crosslinking formed between hydrophilic domains of tropoelastin give rise to the water-insoluble
networks of elastin. 75% of the amino acid residues are hydrophobic in elastin such as Gly, Val
and Ala.
48
Coacervation is an important process to change the phase of tropoelastin under the
control of temperature. Below the transition temperature (Tt), tropoelastin is soluble in aqueous
solutions; however, when above the Tt, tropoelastin aggregates into insoluble fibrillar
structures
49
.

Based on the features and structure of tropoelastin, ELPs were developed. ELPs are repetitive
polypeptides of pentamers with the sequence of (Val-Pro-Gly-Xaa-Gly)n. Xaa represents the
guest residue and n is the number of repeating units of pentameric sequences. Above their Tt,
ELPs aggregate and precipitate out of solution.
50
And for different ELPs, transition temperatures
11
differ, mainly depending on the nature of the guest residue, and how many numbers of unit
repeats.
ELPs have a wide range of applications. They can be used for protein purification by binding to
the functional group that are conjugated to the target protein. Above Tt, the ELP-protein forms
spherical clumps can be physically separated from any other extraneous protein in solution.
51

ELPs can also be used in cancer therapy. ELP-drug complexes can better target tumor cells
through the enhanced permeability and retention (EPR) effect of nanoparticles, and these
nanoparticles can also hide the functional group in the spherical aggregate from detection by
tumor cells.
52
In addition, the brittle gel formed by high concentration of ELPs with their tunable
structure and mechanics can be used to assemble a regeneration scaffold, which is a significant
advance in complex tissue regeneration
53
.

Figure 4. Structure of ELP fused to pro-Caspase-1

In order to further control the activation of IL-1β and characterize the mechanism of cytokine
secretion, we designed the fusion protein in which ELPs are fused to the N-terminus of pro-
Caspase-1. Our hypothesis is that this fusion protein might able to carry out the auto-proteolysis
process of pro-Caspase-1 and activate pro-IL-1β in absence of inflammasomes by increasing the
local concentration of pro-Caspase-1 through the temperature-controlled aggregation of ELPs.
This thesis describes the process of creating the ELP-Caspase-1 fusion protein by cloning
methods and the attempt to express this fusion protein in THP-1 and HEK-293T cell lines.
12
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials
The pCl-Caspase-1 plasmid was purchased from Addgene (Watertown, MA) and the
oligonucleotide for inserting an AfeI restriction digestion site on pCl-Caspase1 plasmid
(sequenced 5’-GGGAATTCGACAGCGCTGTCGAATTCCC-3’) was obtained from Integrated
DNA Technologies, Inc. (Coralville, IA). The pET25b-V72, pET25b-V96 and pET25b-A96
plasmids were offered by Dr. J. Andrew MacKay’s lab. Terrific Broth Powder was purchased
from Hardy Diagnostics (Santa Maria, CA). The agarose LE powder was obtained from Laguna
Scientific (Laguna Niguel, CA). All of the restriction enzymes including EcoRI @HF, AfeI,
AcuI, NdeI, HINDIII @HF, Antarctic Phosphatase, T4 DNA Ligase and Klenow fragment were
purchased, together with corresponding buffers, from New England Biolabs® Inc. (Ipswich,
MA). All the kits for plasmid Miniprep, Maxiprep and Gel Extraction were ordered from
QIAGEN (Hilden, Germany).

The RPMI 1640 medium with L-glutamine, the Dulbecco’s Modification of Eagle’s Medium
(DMEM) with 4.5g/L glucose and Penicillin. Streptomycin was obtained from Corningâ
(Corning, NY). Dulbecco’s Phosphate Buffered Saline (DPBS), Opti-MEMä reduced serum
medium, 0.25% Trypsin-EDTA, fetal bovine serum (FBS), and the Lipofectamine 3000 reagent
were all purchased from ThermoFisher Scientific (Waltham, MA).

The protease inhibitor cocktail was obtained from MedChemExpress (Monmouth Junction, NJ).
The Pierceä BCA Protein Assay Kit and the PageRulerä prestained protein ladder were
purchased from ThermoFisher Scientific. The anti-CASP1 antibody was purchased from Cell
13
Signaling Technology, Inc. (Danvers, MA). Finally, the WesternBright ECL HRP substrate was
from Advansta Inc. (San Jose, CA).

2.2 Solutions and Reagents
Cloning
Terrific Broth Media
51.6g Terrific Broth powder
1mL 1000X Carbenicillin antibiotic
QS 1000mL Distilled water
Terrific Broth Agar plate
25.8g Terrific Broth powder
8.5g Agar powder
QS 500mL Distilled water
500uL 1000X Carbenicillin antibiotic
1% Agarose Gel
1g Agarose powder
QS 100mL distilled H2O
5uL SYBR Safe
1.5%Agarose Gel
0.75g Agarose powder
QS 50mL distilled H2O
2.5uL SYBR Safe

Cell Culture
THP-1 complete growth medium
450mL RPMI 1640 medium
50mL FBS
5mL 5000U/mL Penicillin. Streptomycin
HEK293T complete growth medium
450 DMEM medium
50mL FBS
5mL 100X L-glutamine
10% FBS RPMI 1640 medium
45mL RPMI 1640 medium
5mL FBS
10% FBS DMEM medium
45mL DMEM medium
5mL FBS
1% FBS RPMI 1640 medium 1% FBS DMEM medium
14
50mL RPMI 1640 medium
0.5 FBS
50mL DMEM medium
0.5mL FBS

Western Blot

10% Running Gel
4.0mL H2O
2.5mL 4X Lower Buffer (1.5M Tris×HCl)
3.4mL Acrylamide monomer
100uL 10%SDS
40uL 10%APS
8uL TEMED
4% Stacking Gel
2.18mL H2O
1.25mL 4X Stacking Buffer (0.5M Tris×HCl)
533uL Acrylamide monomer
40uL 10%SDS
40uL 10%APS
8uL TEMED
Tank (Running) Buffer
0.5g Tris Base
14.4g Glycine
10mL 10%SDS
QS 1000mL H2O
10X Transfer Buffer
144g Glycine
30.2g Tris Base
QS 1000mL H2O
10X TBS buffer
24g Tris Base
88g NaCl
QS 1000mL H2O
Adjust pH to 7.6 with 12N HCl
1X TBST
100uL 10X TBS buffer
1mL Tween 20
QS 1000mL H2O
Blocking Buffer
5g BSA or non-fat milk
QS 100mL 1X TBST

2.3 Methods
2.3.1 Create ELP-CASP1 Fusion Plasmids by Cloning Methods
Preparation of CASP1 plasmid stock
Use a sterilized pipette tip to streak bacteria onto carbenicillin agar plates, use fresh sterilized
pipette tips to drag through to produce streak #1 and spread over the plate to create streak #2 and
#3. Incubate the plate overnight at 37℃. On the next day, use pipette tip to pick one colony and
inoculate into 5mL TB media. Incubate at 37℃ and shake at 250 RPM overnight. Take 2mL of
the colony and add it into 250mL TB media and incubate at 37℃ at 250 RPM overnight. Add
glycerol to the colony to a 1:1 ratio, and mix well. Put the mixed colony into 1.5 mL cryovial
tubes and store at -80℃. Extract CASP1 plasmid from the 250mL media by Maxi-Prep following
the QIAGEN protocol.

Preparation and Annealing of Oligonucleotides
Centrifuge the tube for 10 seconds before opening to ensure the oligonucleotide is at the bottom
of the tube. Resuspend the oligonucleotide in nuclease-free water (NF H2O) to a concentration of
100 µM. Dilute five-fold with NF H2O. Heat at 94℃ for 2 minutes. The final concentration of
the annealed oligonucleotide is 10 µM. Remove from heat, cool to room temperature, and store
at -20℃.

Insert the Oligonucleotide at the N-terminus of CASP1 (named oligo-CASP1 Plasmids)
Digest CASP1 plasmid with EcoRI @HF and Antarctic Phosphatase in 50 µL reaction buffer
diluted with water. Incubate at 37℃ for one hour. Deactivate the enzyme by heating the reaction
at 65℃ for 20 minutes. Mix the digesting solution with 6X loading dye. Load samples and 1 kb
16
ladder into 100mL 1% agarose gel. Run the gel at 100V for 40 minutes. Cut the bands at 5.2 kb
and extract the DNA through the Gel-Extraction method, following the QIAGEN protocol.
Digest oligonucleotide also by EcoRI @HF in 20 µL reaction buffer. Incubate at 37℃ for 1 hour
and inactivate the reaction at 65℃ for 20 minutes. Obtain the digested oligonucleotide by a PCR
purification method, following the QIAGEN protocol. Ligate the digested CASP1 plasmid and
oligonucleotide with T4 DNA ligase at the different molar ratios (insert to vector) of 10:1, 5:1
and 7:1. Incubate at 16℃ overnight. Stop the reaction by heating at 65℃ for 10 minutes.

Verification of Oligonucleotide Insertion
Transform the ligated plasmids into TOP10 chemically competent E. coli following the One-
Shot protocol. Spread transformed E. coli on carbenicillin positive agar plates. Invert the plates
and incubate overnight at 37℃. Pick the colonies and inoculate in 5mL TB media at 37℃ and
shaking at 250 RPM overnight. Isolate the DNA by Qiagen MiniPrep kit. Digest the isolated
plasmids with AfeI enzyme and compare them to those not being digested by running an agarose
DNA gel. Secondly, send the samples to GENEWIZ, Inc. for Sanger sequencing and select the
CMV promoter as the primer.

Insert ELPs into oligo-CASP1 Plasmids
Digest oligo-CASP1 plasmid with AfeI enzyme and Antarctic Phosphatase enzyme. Purify the
digested plasmid from a 1% agarose gel by the gel extraction method. Cut V72, V96, and A96
(ELPs) plasmids with both NdeI and AcuI. Separate the 1.1 kb to 1.4 kb ELPs fragments from
the 4 kb plasmid backbones by running them through a 1.5% agarose gel. Remove the 3’
overhangs and fill in 5’ overhangs to create blunt ends by using Klenow (Large) Fragment. Insert
17
the three ELP fragments into the newly created AfeI site in oligo-CASP1 plasmids with the
molar ratio of 3:1 (insert: vector) in different reactions.

Verification of ELP Ligation
Amplify the ligated plasmids according to the steps of E. coli transformation, incubating on
carbenicillin positive agar plates, and Qiagen MiniPrep extraction. For the first round
verification, digest the purified DNA with HinDIII @HF, and check the size based on a DNA
agarose gel. Send the samples of the correct size for Sanger sequencing in both forward and
reverse direction using the CMV promoter and 5’-ctccatctcttccttgttcagc-3’ primers.

2.3.2 ELP-CASP1 expression
Cell Recovery
Remove the cryovial containing THP-1 or HEK-293T cells from liquid nitrogen storage and
thaw in the 37℃ water bath for 2 minutes. Spray 70% ethanol to decontaminate the vial. Transfer
the contents into centrifuge tubes and dilute with 9mL complete growth medium. Centrifuge for
3 minutes at 900 RPM. Resuspend the cell pellet with 10mL complete growth medium and
transfer to a 10cm cell culture dish. Incubate at 37℃ in 5% CO2 air for 2-3 days to wait for the
cell growth.

Cell Culture
THP-1 cells are maintained in the RPMI 1640 medium supplemented with 10% FBS and 5%
antibiotic (Penicillin Streptomycin). Cells are plated in 10cm cell culture dishes at the density of
2-4 x 10
5
cells/mL and incubated at 37℃ in an air atmosphere with 5% CO2. Passage cells every
18
2 -3 days when the density reaches 8 x 10
5
cells/mL. Do not let the density to reach levels higher
than 1 x 10
6
cell/mL. Since THP-1 is a suspension cell line, centrifugation and resuspension are
optional during subculture.

HEK293T cells are maintained in the high glucose DMEM medium supplemented with 10%
FBS and 1% L-glutamine. Subculture the cells when it reaches 80%-90% confluency. Remove
the culture medium and rinse the cell with 3mL DPBS. Add 2mL of Trypsin and incubate at 37℃
for 3 minutes. Add 6mL of growth medium and aspirate cells. Centrifuge at 900 RPM for 3
minutes and discard the supernatant. Resuspend cell pellet with complement growth medium and
plate into 10cm cell culture dishes. Passage the cell every 2-3 days with a subcultivation ratio of
1:5.

Transfection
Two days before transfection, seed 5 x 10
5
cells/well HEK293T cells in 2mL complement
growth medium in 6-well plates, pre-coated with poly-D-lysine to help cells adhere to plates. On
the day of transfection, plate different amounts of THP-1 cells per well in 2mL of 10%,1%, or no
FBS-added RPMI 1640 medium without antibiotics. Replace the old medium with 10%, 1%, or
no FBS-containing DMEM medium for HEK293T cells. Use Lipofectamine 3000 as the
transfection reagent. Prepare DNA-lipid complexes by mixing 5 µL of Lipofectamine 3000
reagent, 5 µL of P3000 reagent and 2.5ug of plasmid DNA, and incubating in 250 µL of Opti-
MEM medium at room temperature for 15 minutes for every well. Different plasmids are
transfected into both THP-1 and HEK293T cells. Incubate for 48 hours in 5% CO2 air at different
19
temperatures of 37℃ and 32℃ respectively to investigate whether the temperature influences the
expression and detection of CASP-ELP fusion protein.

Lysis of THP-1 and HEK293T cells
Adherent HEK293T cells: Remove the culture medium. Wash cells twice with cold PBS. Add
200 µL of cold radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease
inhibitor cocktail which consists the inhibitors of serine, cysteine, aspartic proteases and
aminopeptidase. Incubate on ice for five minutes. Gather cells by cell scraper. Transfer lysate
into microcentrifuge tubes and centrifuge at 1000 RPM for 5 minutes. Store supernatant at -20℃.

Suspension THP-1 cells: Centrifuge cells at 900 RPM for 3 minutes, and discard the supernatant
(culture medium). Add 200 µL protease inhibitor-containing RIPA buffer. Shake on ice for 15
minutes. Centrifuge again with the same parameters as above, and store the supernatant at -20℃ .

BCA Protein Assay
Prepare standard solutions A to I with different concentrations using BSA stock; follow the
Pierce BCA Protein Assay protocol. Add 25 µL of standards and unknown samples into each
well of 96-well plate. Repeat adding all the samples and standards twice to ensure accuracy. Add
200 µL of working reagent (mix component A and B in the ratio of 50:1) into each well. Mix
well. Incubate at 37℃ for 30 minutes. Cool plate to room temperature, and remove the cap.
Measure the absorbance at 562 nm using Microplate Manager Software.

20
Western Blot
Prepare the 10% Lower Gel and 4% Stacking Gel first. Add 5X Laemmli sample buffer to
protein samples. Boil cell lysate at 95℃ for 5 minutes to denature proteins. Load samples and the
PageRulerä prestained protein ladder into different wells. Run at 80V for 1 hour to better
compress the bands and increase the voltage to 100 or 110V and run for 1 to 2 hours. Activate
the PVDF membrane in methanol for 1 minute and rinse with transfer buffer. Prepare the transfer
stack in the sequence of cathode core, sponge pad, filter paper, gel, PVDF membrane, filter paper,
sponge pad and anode core. Transfer overnight at 20V or 300mA for 90 minutes.

Block the membrane for 1 hour at room temperature in blocking buffer. Rinse the membrane in
TBST three times, 10 minutes each. Incubate the membrane with 1000 times diluted anti-CASP1
antibody in 1% BSA in TBST with 0.01% NaN3 overnight at 4℃. Wash the membrane in TBST
3 times, 10 minutes each. Incubate the membrane with 10000 times diluted anti-rabbit HRP-
conjugated secondary antibody in blocking buffer for 1 hour at room temperature. Wash the
membrane 3 times, 10 minutes each in TBST.

For antibody detection, incubate the membrane in 1:1 mixed WesternBright ECL HRP substrate
at room temperature for 1 minute. Acquire the chemiluminescent image and colorimetric
detection using the BIO-RAD system.

21
CHAPTER THREE: RESULTS
3.1 Creation of ELP-CASP1 Plasmids
Oligonucleotide Insertion

Figure 5. Size of EcoRI @ HF D igested pC l-caspase1 Plasmids.
The four boxed bands represent the EcoRI digested 5226bp pCl-Caspase1 plasmids.

EcoRI digested pCl-Caspase1 (plasmid with Caspase-1 gene inserted in the mammaline
expressed pCl vector backbone) plasmids were loaded into four separate wells, with 15 µL in
each well and run in a 1% agarose gel at 100V for 40 minutes. All four bands were located
between the 5 kb and 6 kb ladder bands which were consistent with the 5.2Kb size of the pCl-
Capsase1 plasmids.

5.2kb
22

Figure 6. Positive E.coli colonies of oligo-CASP1 plasmids.
The insert to vector molar ratio of plate 1, 2 and 3 are 10:1, 7:1 and 5:1 respectively.

pCl-Caspase1 (CASP1) plasmids were extracted from the bands cut from the agarose gel.
Ligation of the oligonucleotide (sequenced 5’-GGGAATTCGACAGCGCTGTCGAATTCCC-3’)
and CASP1 plasmids was carried out in three different molar ratios 10:1, 7:1 and 5:1 due to the
small size of the insert fragment. After ligation and transformation, the E. coli were spread onto
the carbenicillin positive agar plates. On the next day, there were no colonies on Plate 1; 12
colonies on Plate 2; and, 10 colonies on Plate 3, consistent with the possibility that the 10:1 ratio
was too high for the plasmid ligation, and the 5:1 ratio was enough for successful insertion of the
short oligonucleotide.

Plate 1 (10：1） Plate 2 (7：1） Plate 3 (5：1）
23

Figure 7. AfeI verification of oligonucleotide insertion.
7 groups of randomly selected samples were loaded into 14 wells in total. Lane 1,3,5,7,9,11 and 13 were AfeI enzyme digested
plasmids, lane 2,4,6,8,10,12 and 14 were non-digested plasmids and lane 15 was original pCl-capase1 plasmid.

All of the 22 colonies were picked and inoculated in 15ml culture tubes to extract plasmid DNA
from them. 7 samples were selected randomly and digested by AfeI enzyme. Both the digested
samples and non-digested samples were loaded to compare the size of the fragments with that of
the original pCl-caspase1 plasmid. Since the circular plasmid usually runs faster than the
linearized plasmid, the bands of non-digested samples, original pCl-caspase1, and those samples
without oligonucleotide successfully inserted would migrate at the position which was smaller
than 5 kb. However, for those samples with oligonucleotide successfully inserted, AfeI would
digest them into linear plasmids as the oligonucleotide contained an AfeI site, which was not
present in the original parent plasmid.

In Fig. 7, samples 2, 3, 4, 6 and 7 showed different band sizes between digested and non-digested
groups. Digested bands were all about 5 kb, and non-digested bands were about 4 kb which was
a size consistent with the original pCl-caspase1 plasmid band. In that case, the insertion of
samples 2, 3, 4, 6 and 7 was considered to be successful. Those samples were then sent for
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample 1 2 3 4 5 6 7
4 kb
5 kb
24
sequencing, wih the CMV promoter as the primer. The sequencing result further confirmed that
the ligation was successful.

ELPs Insertion

Figure 8. AcuI and NdeI digested ELPs plasmids.
pET25b-ELP plasmids were digested by AcuI and NdeI together to cut the ELP fragment off. The 1.0 kb band in lane 1 represent
V72 fragment and 1.5 kb bands in lane 2 and lane3 represent the V96 and A96 fragments.

Since we designed the ELPs to ligate to the N terminus of pro-Caspase1, AcuI and NdeI were
used to cut the V72, V96 and A96 fragments from the respective pET25b-ELP plasmids and to
remove concomitantly the stop codon. The V72 fragment was 1086 bp in length, and the V96
and A96 fragments were both 1.4 kb in length. The three bands were excised, and DNA was
extracted from them.

V72 V96 A96
1.5 kb
1.0 kb
25

Figure 9. Positive E.coli colonies of ELP-CASP1 plasmids .

The ligation was carried out at the molar ratio of 3:1 (insert to vector) for all three ELPs. After
ligation and transformation, the E. coli were spread onto the carbenicillin positive agar plates.
There was only one colony each with the V96 and A96 insertions, and no colony on the plate
with the V72 insertion. Thus, ligation of V72 to pro-Caspase1 did not appear to succeed.

Figure 10. HinDIII @HF restriction digest to verify ELP insert ligation.
After digested by HinDIII @HF, there were two bands located at about 2.0 kb and 4.0 kb for both A96-CASP1 and V96-CASP1
plasmids.

HinDIII @HF was used to verify the ligation of either V96 or A96 to pro-Caspase1. There were
two cutting sites for HinDIII on the oligo-CASP1 plasmid, and the insertion position of ELPs
V96-CASP1 A96-CASP1
4.0 kb
2.0 kb
V96 A96

26
was in-between the two HinDIII digestion sites. When digested by HinDIII, successfully ligated
plasmids would then be split into two fragments of 2.1 kb and 4.5 kb in length, and the oligo-
CASP1 plasmid would be split into two fragments of 0.7 kb and 4.5 kb in length. After running a
DNA agarose gel, both the V96 and A96 ligated plasmids showed two bands at about 2.1 kb and
4.5 kb which indicated that the insertion of ELPs was successful.

However, the ELP fragments were digested by Klenow fragments to create blunt ends, and the
ligations were blunt-end ligations. Further sequencing was needed to make sure the insertion
occurred in the correct direction. The CMV promoter was used as the primer for the forward
sequencing. And the primer used for reverse sequencing was an oligonucleotide designed to
match the “bottom strand”. The sequence of this oligonucleotide was 5’-ctccatctcttccttgttcagc-3’.
The sequencing result indicated that both the A96-CASP1 and V96-CASP1 were in-frame, and
could therefore be used in further experiments.

3.2 Protein Expression
Since CASP1 is a component of inflammasomes, and the final objective of this project is to
characterize the secretion of cytokine IL-1β, I decided to use the THP-1, the white blood cell line
which can express endogenous inflammasomes and IL-1β, to express the fusion protein.

27

Figure 11. Western blot to determine the expression of ELP-CASP1 fusion protein in THP-1 cells grown in 1% FBS RPMI 1640 medium.
To detect the target ELP-CASP1 fusion protein, anti-CASP1 antibody was used as the primary antibody to probe the membrane and the
secondary antibody was the anti-rabbit HRP-conjugated secondary antibody. This figure is the chemiluminescent image of the
western blot. Lane 1: THP-1 treated with 2.5 µl of Lipofectamine 3000 reagent without adding any plasmids. Lane 2 and lane 3:
THP1 transfected with V96-CASP1 plasmids using 2.5 µL and 7.5 µL of Lipofectamine 3000 reagent, respectively. Lane 4:
THP-1 treated with 7.5 µl of Lipofectamine 3000 reagent without adding any plasmids. Lane 5 and lane 6: THP1 transfected with
A96-CASP1 plasmids using 2.5 µL and 7.5 µL of Lipofectamine 3000 reagent, respectively.

For the first set of transfections, I used two different doses of Lipofectamine 3000 reagent
according to the ThermoFisher protocol. The result showed that there was no apparent difference
when using different doses of Lipofectamine 3000 reagent, and thus in subsequent experiments I
used the average of the two doses, which was 5 µL of Lipofectamine 3000 reagent. By using the
anti-CASP1 and anti-ELP as the primary antibodies to detect the ELP-CASP fusion proteins, I
obtained the result showed in Fig. 11. All of the six lanes only had one band at the same position
of about 45 kDa, which was consistent with the molecular weight of endogenous pro-Caspase1
protein in THP-1 cells. From Fig. 11, we conclude that the ELP-CASP1 fusion protein was not
successfully expressed in THP-1 cells.
MW
(kDa)
180
130
100
70
55

40

35
25

15
Lane 1 2 3 4 5 6
28

The problem might have been related to the low transfection efficacy, and/or the failed
transcription or translation process of ELP-CASP1 fusion protein. To investigate if the
transfection could be successful. I used HEK293 and HEK293T cells as they cannot express
endogenous Caspase-1 or any inflammasome-related proteins.

Figure 12. Western blot to determine the expression of ELP-CASP1 fusion protein in HEK293 cells grown in 1% FBS DMEM medium.
Anti-CASP1 antibody and the anti-rabbit HRP-conjugated secondary antibody were used to detect the ELP-CASP1 fusion
protein. This figure is the chemiluminescent image of the western blot. Lane 1: HEK293 cells treated with 5 µL of Lipofectamine
3000 reagent alone. Lane 2: HEK293 cells transfected with 2.5 µg of oligo-CASP1 plasmid. Lane 3 and lane 4: HEK293
transfected with 2.5 µg of V96 and A96 plasmids, respectively. Lane 5 and lane 6: HEK293 transfected with 2.5 µg of V96-
CASP1 and A96-CASP1 plasmids, respectively.

In this experiment, I transfected the CASP-oligo alone, ELPs alone, and ELP ligated CASP1
plasmids into HEK293 cells to compare them to cells treated only with transfection reagent.
After incubated with the anti-CAP1 antibody, only Lane 2, showing a sample from cells
transfected with the oligo-CASP1 plasmid alone had one band around 35 kDa. Based on the
activation and deactivation process of Caspase-1, two pro-caspase1 molecules form a homodimer,
then the IDL linker is cleaved to form the transient p33/p10 active form, and then the p20/p10
Lane 1 2 3 4 5 6
MW
(kDa)
180
130
100
70
55

40

35
25

15
29
species are generated from the p33/p10 active form by self-cleavage. And, according to some
papers, a high local concentration of Caspase1 can also lead to its activation. The Lane 2 band
proved that the transfection is successful because HEK293 cells do not express endogenous
Caspase-1. However, the size of the band was not 45 kDa but 35 kDa. Based on the potential
dimeric species of Caspase-1 (Fig.3B), this might be because the expressed oligo-CASP1 formed
homodimer automatically at high local concentration, and the small subunit was cleaved off. To
verify the 35 kDa represent the p33 form of dimeric Caspase-1, anti-Caspase-1 p10 can be used
in western blot as the primary antibody. If there is no band appear at 35 kDa when using anti-
Caspase-1 p10 but do have band appear at 35 kDa when using anti-CASP1 antibody, the p33
form can be confirmed. Thus, the HEK293 cell line seemed suitable to investigate the expression
of CASP1 plasmids.

Figure 13. Western blot to determine the expression of ELP-Casp1 fusion proteins in HE K293T cells grown in 10% FBS RP MI 1640
medium.
Lane 1 to lane 4 were incubated at 37℃, lane 5 to lane 8 were incubated at 32℃, both in air with 5% CO 2. Lane 1 and lane 5 were
transfected with a plasmid with cDNA encoding wild type CASP1. Lane 2, 6 were transfected with 2.5 µg of oligo-CASP1
MW
(kDa)
180
130
100
70
55

40

35
25

15
Lane 1 2 3 4 5 6 7 8
30
plasmids. Lane 3, 4, 7 and 8 are cell lysates of HEK293T cells transfected with 2.5µg of V96-CASP1 and A96-CASP1 plasmids,
respectively.

From the western blot, the bands of oligo-CASP1 was lighter than the bands of wild type
Caspase-1 at 37℃. At 32℃, there was no difference between the bands of oligo-CASP1 and that
of Caspase-1. At 37℃, both wild type CASP1 and oligo-CASP1 had two bands at 45KDa and
35KDa that may represent the original form of pro-Caspase1 and active p33 form. The active
p33 form can also be confirmed by using anti-Caspase-1 p10 antibody as the primary antibody
when doing western blot as mentioned before. At 32℃, in addition to the 45 kDa and 35 kDa
bands observed in cells incubated at 37°C, there was a band at around 15 kDa in both Lane 5 and
Lane 6. Because the anti-CASP1 antibody binds to the epitope in p20 of Caspase-1, the 15 kDa
band was likely to represent the inactivated free p20 subunit of Caspase-1. Unfortunately,
however, for the V96 and A96 ligated Caspase-1, there were still no bands that appeared in
Lanes 3 and 4 and Lanes 7 and 8, suggesting that the ELP-CASP1 fusion protein was not
successfully expressed.

According to some papers, the percentage of FBS in the culture medium also influences the
efficacy of transfection and protein expression, especially the high level of FBS usually inhibit
the expression of transfected protein. So, I also tried to incubate the HEK293T cells after
transfection in 1% FBS growth medium instead of 10% FBS growth medium to see if there is
any difference in either the efficacy of transfection or protein expression. Based on the result that
wild type Caspase-1 and oligo-CASP1 did not show a significant difference in expression in
HEK293T cells, only wild type CASP1 was used this time.

31

Figure 14. Western blot to determine the expression of ELP-Casp1 fusion proteins in HEK293T cultured in 1% FBS RPMI 1640 medium.
Lane 1 to Lane 3 were incubated at 37℃, Lane 4 to Lane 6 were incubated at 32℃, and all were incubated under 5% CO 2. Lanes
1 and 4 were transfected with wild type CASP1 plasmids. Lanes 2 and 5 were transfected with the V96-CASP1 plasmid. Lanes 3
and 6 were transfected with the A96-CASP1 plasmid.

Poly-D-lysine was not available in the lab when seeding HEK293T cells. As a result, the cell did
not grow as well as it did in previous experiments. When loading apparently the same amount of
protein in each well, the bands can still be seen in this image (Fig. 14) although not clearly as
before. In the cell lysate of cells incubated at 37℃, Lane 1 had three bands at 45 kDa, 40 kDa
and 35 kDa; these bands may represent the pro-Caspase1 (45kDa band) and the active p33 form
(35 kDa band). Surprisingly, Lane 4 also showed two bands at about 100 kDa and 40 kDa. Since
all the cells were seeded into the same 6 well plate and were treated by the same method at the
same time, and the bands in Lane 4 were almost at the same position as in Lane 5 in Fig. 13, the
appearance of the 100 kDa and 40kDa bands were not likely to be artefacts. The 40 kDa band
might represent the pro-Caspase1 that typically migrates at 45 kDa or p33 form of active
Caspase-1that typically migrates at 35 kDa which need to be further verified. The 100 kDa band
MW
(kDa)
180
130
100
70
55

40

35
25

15
Lane 1 2 3 4 5 6
32
was consistent with the predicted molecular mass of A96-CASP1 fusion protein, which was
predicted to be about 95 kDa. From Lane 4 to Lane 6, only the group of cells transfected with
the wild type CASP1 showed bands at different positions, possibly representing different forms
of Caspase-1 as also observed in reference 47. Also, the A96-CASP1 fusion protein seemed to be
expressed at 37℃ in cells cultured in the 1%FBS medium, although it was not expressed in in
high amounts.

33
CHAPTER FOUR: DISCUSSION
IL-1β plays important roles in innate immunity and involved in many immune diseases. By
understanding the secretion mechanism of IL-1β, it will be of great benefit for the treatment of
diseases such as rheumatoid arthritis, atherosclerosis, asthma, and cancers. In this project, to
make it easier to control the activation of IL-1β, the approach was to ligate different ELPs, which
can aggregate together in a temperature-dependent fashion to the N-terminus of pro-Caspase1 to
control perhaps more precisely the activation of caspase-1, and, thus, the initiation of secretion of
IL-1β to be able to characterize its mechanism of secretion.

The first part of the project was the plasmid construction using cloning methods. There were two
steps to insert an oligonucleotide to only create an AfeI restriction digestion site and then insert
various ELPs into the created site, shown in Fig. 5 to Fig. 10. The second part of the project was
to express fusion proteins of the various ELPs fused to pro-Caspase1, and express them in white
blood cells or other human cell lines to see if the construct can be expressed, as shown from
Fig.11 to Fig. 14.

The plasmid construction process of V96 and A96 fused to pro-Caspase1 seemingly went very
well. The ligation succeeded on the first try. However, the ligation of V72 to the pro-Caspase1
did not seem to succeed after several attempts. The first problem was that the V72 fragment was
difficult to be separated from the pET25b plasmid backbone. Since there were three digestion
sites of AcuI in the pET25b-V72 plasmid, four fragments would be generated after digestion.
Among them, the V72 fragment was 1086bp in length, two other short fragments, which were
similar in length to the V72 fragment, were 996bp and 1012bp in length, respectively, and finally,
34
a 3.4 kb fragment. Because the fragment lengths were too close in size, a 1.5% agarose gel could
not separate through electrophoresis the V72 fragment from the other two short fragments. As
shown in Fig. 7, except for the 3.4 kb band, the V72 lane had only one band of 1 kb, but in the
V96 and A96 lanes there were two bands of 1.4 kb and 1.0 kb, representing the V96 and A96
fragments and two short fragments of the pET25b plasmid backbone, respectively.

To avoid this problem, I also tried a two-round digestion. First using AcuI to digest the plasmid
and extract the large 5 kb fragment containing the V72 sequence and then using NdeI to digest
the extracted large fragment, since the shorter fragment of 1 kb is the fragment encoding V72
that was needed. However, another problem arose, because the efficacy of the gel extraction was
not high, and this method required multiple rounds of gel extractions and PCR purification of
DNA. Despite these enhancements, there was still a failure to obtain sufficient quantities of V72
DNA. This problem also occurred when I tried to use other enzymes for the two rounds of
digestion.

To rule out that the V72-encoding plasmid sequence was incorrect and that the digestion could
be performed correctly, I also performed gene sequencing on the V72 plasmid. The sequencing
results showed that the plasmid sequence was accurate. To ensure the quality of the plasmid, I
also transformed the plasmid into E. coli and picked colonies growing on antibiotic-containing
agar plates for further plasmid amplification and extraction. However, after repeating the above-
mentioned digestion and ligation methods multiple times, V72 was still not successfully ligated
into the pCl-caspase1 plasmid. In order to ensure the progress of the remaining part of the project,
35
I stopped the construction of this plasmid and only used the successfully ligated V96-CASP1 and
A96-CASP plasmids during the subsequent protein expression experiments.

For the selection of a cell line, THP-1 cells were my first choice. Since I am targeting Caspase-1
and the upstream and downstream processes mediated by inflammasomes and IL-1β, immune
cells such as THP-1 cells, a white blood cell line which can express all of the components of
inflammasomes, IL-1β, and also GSDMD appeared to be well suited for this project.

However, as shown in Fig. 10, the high level of endogenous Caspase-1 appeared to impact the
detection of Caspase-1 and the expression of the fusion protein, especially when the expression
level of the constructs within transfected plasmids was low or not expressed at all. And when the
Caspase-1 level was too high in the cell, it would lead to apoptosis, which also appeared to affect
the detection of expressed protein. So in subsequent experiments, I changed to HEK293 and
HEK293T cells, the general human cell lines that did not express endogenous Caspase-1 and IL-
1β, to determine if the plasmids were suitable for expression of ELP-CASP1 fusion proteins.
And more control groups, such as expression plasmids for wild-type CASP1, the oligo-CASP1,
and ELPs alone, were incorporated into the analysis to further explore transcription and
translation of these protein constructs.

In experiments with HEK293 and HEK293T cells, I also had used different culture methods,
such as different concentrations of FBS supplemented into the culture medium and different
temperatures. I also continued the use of multiple expression plasmids, including those for the
wild-type CASP1, oligo-CASP1, ELPs alone, and ELP fused to CASP1. Comparing the results
36
obtained, as shown in Fig. 11 to Fig. 13, when culturing at lower temperature, more protein
could be detected through western blot. This result was consistent with other studies showing
that protein expression level is increased in HEK293 cell by lowering culture temperature
56
.
When using the 1% FBS-supplemented cell culture medium, the protein expression level
decreased especially those cultured at 37℃. Although at a low level, a 100 kDa band in the lane
with the lysates of cells transfected with the A96-CASP1 expression plasmid was detected by
western blot with anti-CASP1 antibody. Based upon the molecular weight and immunoreactivity,
this band was inferred to be the A96-pro-Caspase1 fusion protein. The reason why the cells
cultured at 32℃ showed higher protein expression level but could not express fusion protein was
not clear. Low temperature may have inhibited transcription or translation of a large fusion
protein. This was the first time the expressed fusion protein may have been detected, although at
a low level. The future steps of this project may still be pursued.

Due to the lack of time and outbreak of COVID-19, this project has to be stopped. Much more
work can be done in the future to improve the existing results, including trying more methods to
create a V72-CASP1 fusion protein plasmid and using different cell culture conditions or
treatments to find the best way that results in express high level of fusion protein. Some
experiments that can be tried later are listed below:

1) Use poly-D-lysine to incubate the wells before seeding HEK293T cells. Culture the cells
in 1% FBS-supplemented DMEM medium, and compare the result with the experiment
shown in Fig. 13 to see if poly-D-lysine has impact on cell growth and protein expression.
37
2) Use immunofluorescence staining method targeting the Myc-tag on the C-terminus of
Caspase-1 to detect whether the transfected plasmid is expressed. The Myc-tag
immunofluorescent detection is more sensitive than western blot especially when the
proteins are aggregated together since the aggregated large protein is difficult to migrate
in western blot and will appear as large, long dark bands at the top of the SDS page.
3) Use a Caspase-1 inhibitor to treat cells after transfection. Because of the self-activation
and degradation of Caspase-1 at high local concentrations, these fusion proteins may not
be able to exist long enough after translation which impacts the detection of these fusion
proteins.
38
REFERENCES
1. Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate
immunity. Cell, 124, 783-801.
2. Dinarello, C. A. (1998). Interleukin-1β, Interleukin-18, and the Interleukin-1β Converting
Enzyme a. Ann N Y Acad Sci, 856, 1-11.
3. Ren, K., and Torres, R. (2009). Role of interleukin-1β during pain and
inflammation. Brain research reviews, 60, 57-64.
4. Dinarello, C. A. (1991). Interleukin-1 and interleukin-1 antagonism.
5. Hallegua, D. S., and Weisman, M. H. (2002). Potential therapeutic uses of interleukin 1
receptor antagonists in human diseases. ANN RHEUM DIS, 61, 960-967.
6. Bent, R., Moll, L., Grabbe, S., and Bros, M. (2018). Interleukin-1 Beta—A Friend or Foe
in Malignancies?. Int. J. Mol. Sci., 19, 2155.
7. Mantovani, A., Barajon, I., and Garlanda, C. (2018). IL-1 and IL-1 regulatory pathways
in cancer progression and therapy. Immunol. Rev., 281, 57-61.
8. Kawai, T., and Akira, S. (2007). Signaling to NF-κB by Toll-like receptors. Trends. Mol.
Med., 13, 460-469.
9. Burns, K., Martinon, F., and Tschopp, J. (2003). New insights into the mechanism of IL-
1β maturation. CURR OPIN IMMUNOL, 15, 26-30.
10. Fenton, M. J. (1992). transcriptional and post-transcriptional regulation of interleukin 1
gene expression. Int. Immunopharmacol., 14, 401-411.
11. He, Y., Hara, H., and Núñez, G. (2016). Mechanism and regulation of NLRP3
inflammasome activation. Trends Biochem. Sci., 41, 1012-1021.
12. Sharma, D., and Kanneganti, T. D. (2016). The cell biology of inflammasomes:
Mechanisms of inflammasome activation and regulation. J CELL BIOL, 213, 617-629.
13. Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D.
R., ... and Fitzgerald, K. A. (2009). AIM2 recognizes cytosolic dsDNA and forms a
Caspase-1-activating inflammasome with ASC. Nature, 458, 514-518.
14. Rathinam, V. A., Jiang, Z., Waggoner, S. N., Sharma, S., Cole, L. E., Waggoner, L., ...
and Hornung, V. (2010). The AIM2 inflammasome is essential for host defense against
cytosolic bacteria and DNA viruses. Nat. Immunol., 11, 395.
39
15. Schnappauf, O., Chae, J. J., Kastner, D. L., and Aksentijevich, I. (2019). The Pyrin
Inflammasome in Health and Disease. FRONT IMMUNOL, 10, 1745.
16. Muñoz-Planillo, R., Kuffa, P., Martínez-Colón, G., Smith, B. L., Rajendiran, T. M., and
Núñez, G. (2013). K+ efflux is the common trigger of NLRP3 inflammasome activation
by bacterial toxins and particulate matter. Immunity, 38, 1142-1153.
17. Murakami, T., Ockinger, J., Yu, J., Byles, V., McColl, A., Hofer, A. M., and Horng, T.
(2012). Critical role for calcium mobilization in activation of the NLRP3
inflammasome. PNAS, 109, 11282-11287.
18. Dostert, C., Pétrilli, V., Van Bruggen, R., Steele, C., Mossman, B. T., and Tschopp, J.
(2008). Innate immune activation through Nalp3 inflammasome sensing of asbestos and
silica. Sci, 320, 674-677.
19. Zhou, R., Yazdi, A. S., Menu, P., and Tschopp, J. (2011). A role for mitochondria in
NLRP3 inflammasome activation. Nature, 469, 221-225.
20. Chu, J., Thomas, L. M., Watkins, S. C., Franchi, L., Núñez, G., and Salter, R. D. (2009).
Cholesterol-dependent cytolysins induce rapid release of mature IL-1β from murine
macrophages in a NLRP3 inflammasome and cathepsin B-dependent manner. J. Leukoc.
Biol., 86, 1227-1238.
21. Hornung, V., Bauernfeind, F., Halle, A., Samstad, E. O., Kono, H., Rock, K. L., ... and
Latz, E. (2008). Silica crystals and aluminum salts activate the NALP3 inflammasome
through phagosomal destabilization. Nat. Immunol., 9, 847.
22. Yaron, Jordan. (2015). Ion Flux Regulates Inflammasome Signaling.
10.13140/RG.2.2.32297.60007.
23. Lu, A., Magupalli, V. G., Ruan, J., Yin, Q., Atianand, M. K., Vos, M. R., ... and Egelman,
E. H. (2014). Unified polymerization mechanism for the assembly of ASC-dependent
inflammasomes. Cell, 156, 1193-1206.
24. Mangan, M. S., Olhava, E. J., Roush, W. R., Seidel, H. M., Glick, G. D., and Latz, E.
(2018). Targeting the NLRP3 inflammasome in inflammatory diseases. Nat. Rev. Drug
Discov., 17, 588.
25. Duncan, J. A., Bergstralh, D. T., Wang, Y., Willingham, S. B., Ye, Z., Zimmermann, A.
G., and Ting, J. P. Y. (2007). Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and
requires ATP binding to mediate inflammatory signaling. PNAS, 104, 8041-8046.
40
26. Duncan, J. A., Bergstralh, D. T., Wang, Y., Willingham, S. B., Ye, Z., Zimmermann, A.
G., and Ting, J. P. Y. (2007). Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and
requires ATP binding to mediate inflammatory signaling. PNAS, 104, 8041-8046.
27. Dick, M. S., Sborgi, L., Rühl, S., Hiller, S., and Broz, P. (2016). ASC filament formation
serves as a signal amplification mechanism for inflammasomes. Nat. Commu, 7, 1-13.
28. Viotti, C. (2016). ER to golgi-dependent protein secretion: the conventional pathway.
In Unconventional Protein Secretion (pp. 3-29). Humana Press, New York, NY.
29. Cross, B. C., Sinning, I., Luirink, J., and High, S. (2009). Delivering proteins for export
from the cytosol. Nat. Rev. Mol. Cell Biol., 10, 255-264.
30. Schwartz, T. U. (2007). Origins and evolution of cotranslational transport to the ER.
In Eukaryotic Membranes and Cytoskeleton (pp. 52-60). Springer, New York, NY.
31. Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F., Rich, A., Wolff, S. M., and
Dinarello, C. A. (1984). Nucleotide sequence of human monocyte interleukin 1 precursor
cDNA. PNAS, 81, 7907-7911.
32. Andrei, C., Dazzi, C., Lotti, L., Torrisi, M. R., Chimini, G., and Rubartelli, A. (1999).
The secretory route of the leaderless protein interleukin 1β involves exocytosis of
endolysosome-related vesicles. Mol Cell Biol, 10, 1463-1475.
33. MacKenzie, A., Wilson, H. L., Kiss-Toth, E., Dower, S. K., North, R. A., and Surprenant,
A. (2001). Rapid secretion of interleukin-1β by microvesicle shedding. Immunity, 15,
825-835.
34. Qu, Y., Franchi, L., Nunez, G., and Dubyak, G. R. (2007). Nonclassical IL-1β secretion
stimulated by P2X7 receptors is dependent on inflammasome activation and correlated
with exosome release in murine macrophages. J. Immunol., 179, 1913-1925.
35. Monteleone, M., Stow, J. L., and Schroder, K. (2015). Mechanisms of unconventional
secretion of IL-1 family cytokines. Cytokine, 74, 213-218.
36. Harris, J., Hartman, M., Roche, C., Zeng, S. G., O'Shea, A., Sharp, F. A., ... and Kornfeld,
H. (2011). Autophagy controls IL-1β secretion by targeting pro-IL-1β for
degradation. Int. J. Biol. Chem., 286, 9587-9597.
37. Shirasaki, Y., Yamagishi, M., Suzuki, N., Izawa, K., Nakahara, A., Mizuno, J., ... and
Ohara, O. (2014). Real-time single-cell imaging of protein secretion. Sci. Rep., 4, 4736.
41
38. Shi, J., Zhao, Y., Wang, K., Shi, X., Wang, Y., Huang, H., ... and Shao, F. (2015).
Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell
death. Nature, 526, 660-665.
39. Ding, J., Wang, K., Liu, W., She, Y., Sun, Q., Shi, J., ... and Shao, F. (2016). Pore-
forming activity and structural autoinhibition of the gasdermin family. Nature, 535, 111-
116.
40. Brough, D., Pelegrin, P., and Nickel, W. (2017). An emerging case for membrane pore
formation as a common mechanism for the unconventional secretion of FGF2 and IL-
1β. J Cell Sci, 130, 3197-3202.
41. Black, R. A., Kronheim, S. R., Merriam, J. E., March, C. J., and Hopp, T. P. (1989). A
pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1
beta. Int. J. Biol. Chem., 264, 5323-5326.
42. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura,
M. J., ... and Elliston, K. O. (1992). A novel heterodimeric cysteine protease is required
for interleukin-1βprocessing in monocytes. Nature, 356, 768-774.
43. Boucher, D., Monteleone, M., Coll, R. C., Chen, K. W., Ross, C. M., Teo, J. L., ... and
Yap, A. S. (2018). Caspase-1 self-cleavage is an intrinsic mechanism to terminate
inflammasome activity. J EXP MED, 215, 827-840.
44. Datta, D., McClendon, C. L., Jacobson, M. P., and Wells, J. A. (2013). Substrate and
inhibitor-induced dimerization and cooperativity in Caspase-1 but not caspase-3. Int. J.
Biol. Chem., 288, 9971-9981.
45. Broz, P., von Moltke, J., Jones, J. W., Vance, R. E., and Monack, D. M. (2010).
Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death
and cytokine processing. Cell Host Microbe, 8, 471-483.
46. Cerretti, D. P., Kozlosky, C. J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.
A., ... and Cannizzaro, L. A. (1992). Molecular cloning of the interleukin-1 beta
converting enzyme. Sci., 256, 97-100.
47. Boucher, D., Monteleone, M., Coll, R. C., Chen, K. W., Ross, C. M., Teo, J. L., ... and
Yap, A. S. (2018). Caspase-1 self-cleavage is an intrinsic mechanism to terminate
inflammasome activity. J EXP MED, 215, 827-840.
42
48. Daamen, W. F., Veerkamp, J. H., Van Hest, J. C. M., and Van Kuppevelt, T. H. (2007).
Elastin as a biomaterial for tissue engineering. Biomaterials, 28, 4378-4398.
49. Urry, D. W., Long, M. M., Cox, B. A., Ohnishi, T., Mitchell, L. W., and Jacobs, M.
(1974). The synthetic polypentapeptide of elastin coacervates and forms filamentous
aggregates. Biochimica et Biophysica Acta (BBA)-Protein Structure, 371, 597-602.
50. Shi, P., Lin, Y. A., Pastuszka, M., Cui, H., and MacKay, J. A. (2014). Triggered Sorting
and Co-Assembly of Genetically Engineered Protein Microdomains in the
Cytoplasm. ADV MATER, 26, 449-454.
51. Hassouneh, W., Christensen, T., and Chilkoti, A. (2010). Elastin-like polypeptides as a
purification tag for recombinant proteins. Curr Protoc Protein Sci, 61, 6-11.
52. Saxena, R., and Nanjan, M. J. (2015). Elastin-like polypeptides and their applications in
anticancer drug delivery systems: a review. Drug Deliv., 22, 156-167.
53. Glassman, M. J., Avery, R. K., Khademhosseini, A., and Olsen, B. D. (2016).
Toughening of thermoresponsive arrested networks of elastin-like polypeptides to
engineer cytocompatible tissue scaffolds. Biomacromolecules, 17, 415-426.
54. Dinarello, C. A. (2010). IL-1: Discoveries, controversies and future directions. EUR J
IMMUNOL, 40, 599-606.
55. Krumm, B., Xiang, Y., and Deng, J. (2014). Structural biology of the IL-1 superfamily:
Key cytokines in the regulation of immune and inflammatory responses. PROTEIN
SCI 23, 526-538.
56. Lin, C. Y., Huang, Z., Wen, W., Wu, A., Wang, C., and Niu, L. (2015). Enhancing
protein expression in HEK-293 cells by lowering culture temperature. PloS one, 10.

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Asset Metadata

Creator Wang, Yutong (author)

Core Title Characterization of IL-1β secretion by fusing elastin-like polypeptides to pro-caspase-1

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/27/2020

Defense Date 07/25/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag caspase-1,elastin-like polypeptides,HEK293 cells,inflammasomes,interleukin-1β,OAI-PMH Harvest,THP-1 cells,transfection,western blot

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Okamoto, Curtis (committee chair), Haworth, Ian (committee member), MacKay, Andrew (committee member)

Creator Email wangyuto@usc.edu,wyutong1996@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-341404

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