University of Southern California Dissertations and Theses

Yeast-based nanobody identification for MCEMP1: mast-cell expressed membrane protein 1

(USC Thesis Other)

Yeast-based nanobody identification for MCEMP1: mast-cell expressed membrane protein 1

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

YEAST-BASED NANOBODY IDENTIFICATION FOR MCEMP1:
MAST-CELL EXPRESSED MEMBRANE PROTEIN 1

by

Yu Xiong

A Dissertation Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

December 2022

Copyright 2022 Yu Xiong
ii
Acknowledgements
I would like to express my deepest gratitude to my academic advisor Dr. Jae U. Jung, who
offered me an immersive lab environment and inspired me to explore and solve specific biomedical
problems which are important both to scientific studies and to patients. I would like to extend my
sincere thanks to my mentor Dr. Youn Jung Choi, who not only introduced me all different
experimental skills but also taught me how to make plans for experiments and how to solve
unexpected problems in research. I would like to recognize Dr. James Bowman, who taught me
fluorescence activated cell sorting and all other lab members who always patiently answered my
questions and gave me ideas. Thanks should also go to my defense committees Dr. Omid Akbari,
Dr. Weiming Yuan and Dr. Siyi Chen for reviewing my paper and offering important suggestions.
Special thanks to our program advisor Dr. Joseph R. Landolph, who are also one of the committees,
for reviewing my paper, always encouraging me to keep working on my thesis during the pandemic
and helping me propel the progression of my defense. Lastly, I am also grateful for my parents
Sufang Chen and Weihui Xiong, who were always supportive when I struggled with all the
difficulties throughout this work.

iii
TABLE OF CONTENTS
Acknowledgements ......................................................................................................................... ii
Chapter 1: Introduction ................................................................................................................... 1
Mast Cell as A Therapeutic Target ......................................................................... 1
Mast Cell: Maturation, Activation and Classification ............................................ 3
MCEMP1 Structure and Characteristics ................................................................. 5
Antibodies and Antibody Fragments as Research and Therapeutic Tools ............. 6
Summary of Chapter 1 ............................................................................................ 9
Chapter 2: Large-Scale Protein Production and their Purification ............................................... 11
Introduction ........................................................................................................... 11
MCEMP1 Ectodomain (MCECTO) as an Antigen .............................................. 11
MCECTO Cloning and Transient Gene Expression of 293T Cells ...................... 12
Design of Recombinant Plasmid and MCECTO Purification .............................. 13
Optimization of Cell Culture Routine and Protein Purification............................ 15
Chapter 3: Identification of Nanobodies that Bind to Protein MCECTO ..................................... 24
Introduction ........................................................................................................... 24
Yeast-based Nanobody Selection by Magnetic Cell Sorting (MACS) ................. 25
Yeast-based Nanobody Selection by Fluorescence Activated Cell Sorting (FACS)
............................................................................................................................... 27
Binding Test for Sorted Clones and MCECTO .................................................... 27
Nanobody Sequencing and Preparation for Nanobody Purification in E. coli ..... 29
Chapter 4: Discussion ................................................................................................................... 31
Chapter 5: Materials and Methods ................................................................................................ 33
Cells ...................................................................................................................... 33
Transfection .......................................................................................................... 33
MCECTO Purification .......................................................................................... 34
SDS-PAGE, Coomassie and Western Blot ........................................................... 37
Dialysis and BCA Assay ....................................................................................... 38
iv
Yeast Nanobody Selection .................................................................................... 39
Nanobody Sequencing .......................................................................................... 41
Cloning and Primers ............................................................................................. 42
References ..................................................................................................................................... 44
1
Chapter 1: Introduction
Mast Cell as A Therapeutic Target
Mast cells are hematopoietic tissue-resident granular immune cells on guard at the human
skin and the mucosa of the gut and airways. They execute defensive roles in both innate and
adaptive immunity protecting hosts against parasites, bacteria, and venoms. They also contribute
to repairing damaged structures after injury. However, abundant immune mediators released by
mast-cell granules also make them a source of danger in human body. As far as we know, mast
cells can trigger acute or long-term allergic responses, chronic inflammation, pathogenic tissue
remodeling and can even cause rapid death via anaphylactic shock (Galli & Tsai, 2012). In
consideration of lung health, evidence has shown that their inappropriate and chronic activation
can contribute to the pathophysiology of asthma, pulmonary fibrosis, and pulmonary
hypertension. They are also likely to play a part in chronic obstructive pulmonary disease
(COPD), acute respiratory distress syndrome, and lung cancer (Virk et al., 2016). These
devastating features allow mast cells to be potential therapeutic targets of a variety of human
diseases.

The earliest mast cell-related drug can date back to 1942 when an small-molecule
antihistamine (phenbenzamine) targeting the H1 receptor of histamine was first introduced into
clinical practice for the treatment of a broad range of allergic and other inflammatory reactions
(Leurs et al., 2011). It was even ten years earlier than the discovery that mast cells are the major
tissue source of histamine. While the antihistamines have been the mainstay for mast-cell driven
diseases since their discovery and work well as short-term treatments for mild symptoms, the
approval of an anti-immunoglobulin E (anti-IgE) monoclonal antibody (omalizumab) in the
2
United States in 2003 for treatment of moderate-to-severe allergic asthma in adults and
adolescents aged over 12 years inadequately controlled with inhaled corticosteroids (ICSs), bring
mast cell-targeted therapy into a new era (Chipps et al., 2012). It was later approved for chronic
spontaneous urticaria (CSU) in 2014 as well and it is still the only approved treatment for
antihistamine refractory CSU till today (Maurer et al., 2013). This drug can inhibit mast cell
activation and degranulation via specifically blocking the binding of free IgE to its receptor
FcεRI both on the surface of mast cells and basophils. Other mast cell-targeted drugs that have
been developed recently can also function by blocking specific signal transduction pathways
involved in mast cell activation (for example, BTK), silencing mast cells via inhibitory receptors
(such as Siglec-8) or reducing mast cell numbers and preventing their differentiation by acting on
the mast/stem cell growth factor receptor KIT (Kolkhir et al., 2021).

Based on a preliminary discovery that mast cell-expressed membrane protein 1
(MCEMP1) might be able to enhance KIT-mediated mast cell proliferation and differentiation in
Dr. Jae U. Jung’s laboratory, an experimental tool to activate or inhibit MCEMP1-mediated
signaling was in need for the evaluation of its function. We hypothesized that MCEMP1 might
contribute to KIT activation as an adaptor molecule similar to the linker for activation of T cells
(LAT), as an essential adaptor for FcεRI receptor (Figure 1). LAT positively regulates FcεRI
receptor signaling that induces IgE-mediated mast cell activation and degranulation to release the
various pro-inflammatory mediators (Gilfillan & Tkaczyk, 2006). Although little is known about
MCEMP1 function, there are mounting evidences indicating that MCEMP1 plays a critical role
in allergic and inflammatory lung diseases such as asthma and idiopathic pulmonary fibrosis
(Rastogi et al., 2018; Su et al., 2018; Subrata et al., 2009). The level of MCEMP1 expression is
3
correlated with the severity of lung diseases. These indicate that MCEMP1 plays a critical role in
lung inflammation. Thus, we aimed to develop MCEMP1 inhibitor as a potential therapeutic
strategy for lung inflammatory diseases. Recently a new technique for nanobody discovery
bypasses the llama immunization and instead relies on displaying a synthetic nanobody library
on the surface of yeast cells (McMahon et al., 2018). This study aimed to identify a MCEMP1-
inhibitory nanobody using the yeast surface display system with MCEMP1 ectodomain as an
antigen.

Figure 1: Schematic diagram showing MCEMP1-KIT interaction and LAT-FcεRI interaction in mast cells; Similar
to LAT as an essential adaptor for FcεRI receptor, MCEMP1 is a critical adaptor for KIT receptor that promotes
SCF-mediated mast cell proliferation.

Mast Cell: Maturation, Activation and Classification
Mast cells were first described by the German prolific physician researcher and Nobel
Prize winner, Dr. Paul Ehrlich, in his doctoral thesis in 1878 as “mastzellen” and “granular cells
of the connective tissue,” because of the plump microscopic appearance of these cells and his
discovery of granules in these cells reacting to aniline dyes to give a characteristic change in the
color of staining (Ghably et al., 2015). It was later first demonstrated by Jolly in 1900 that mast
cells derived from bone marrow (Crivellato et al., 2003). Different from most hematopoietic
4
cells leaving bone marrow as an identifiable mature state, mast cells leave bone marrow and
migrate to blood as CD34
+
/CD117
+
progenitor cells, which are less- or non-granulated and are
therefore largely unidentifiable with traditional histochemical staining techniques (Dahlin &
Hallgren, 2015). It is only when mast cell progenitors migrate from blood to peripheral tissues
including skin and mucosal surfaces of eye, respiratory, and gastrointestinal tracts that they
become fully mature and differentiate into considerable subtypes under strict control of micro-
environmental stimuli (Komi et al., 2020). The progression of this maturation relies on KIT
(CD117) activation, a process featured by ligand binding of stem cell factor (SCF) and
subsequent KIT dimerization and auto-phosphorylation (Gilfillan et al., 2011). Besides,
investigations have shown that SCF is critical for the regulation of mast cells in various aspects:
survival, proliferation, migration, maturation, secretion of mediators and IgE-dependent
activation, which makes it one of the most important factors for mast cells (Galli et al., 1993).
Therefore, it is worthwhile to study SCF-KIT activation in mast cell-related health problems.

Distinguished by distinct residing location and histochemical staining characteristics led
by different proteoglycans, rodent mast cells are generally classified into connective tissue mast
cells (CTMC) and mucosal mast cells (MMC) (Aldenborg & Enerbäck, 1988). Different from
rodents, the main classification of human mast cells is according to their distinct contents of
serine proteases and are classified as tryptase/chymase (TC)-positive or T-positive depending on
what they contain, both tryptase and chymase or tryptase only, respectively (Kitamura, 1989).
Nowadays, it is thought that mast cells have much more complexity in terms of their
phenotypes/gene expressions when considering their functional state in inflammatory responses
(Galli et al., 2020).
5

The best-studied activation mechanism of mast cells found in allergic disorders as well as
certain protective immune responses against parasites is achieved by allergen- and IgE-induced
crosslinking of cell surface receptor FcεRI, which leads to the rapid release of a series mediators
including preformed amines and proteases stored in the granules, newly synthesized
proinflammatory lipid mediators and other mediators like growth factors, cytokines and
chemokines (Kalesnikoff & Galli, 2008). Upon allergen binding, IgE-induced gathering of FcεRI
on mast cell surface triggers downstream phosphorylation and activation, which finally brings
about the release of the various mediators. Besides, mast cells can also be activated through other
ligand receptor interactions, which are independent of IgE. These include C3a-C3aR, C5a-C5aR
(CD-88), IgG-FcγRI, and NGF-TRKA, and opioid receptors (Komi et al., 2020).

MCEMP1 Structure and Characteristics
MCEMP1 was identified as a type 2 transmembrane protein on mast cells with its C
terminus extending to the extracellular spaces (Li et al., 2005). From Li’s analysis of its gene
location, it was mapped onto human chromosome 19p13.3 with immune receptor genes-
regulative binding motifs (NF-κB and NF-AT) in the promotor region and with immune-related
neighbor genes (CD23, low affinity receptor for IgE, and FIZZ3, a protein found in
inflammatory zone 3). Based on Li’s research, the mRNA expression levels of MCEMP1
increased and reached a peak when the primary cord blood derived CD34+ cells was cultured
from 1 week to 5 weeks, which gave evidence to the assumption that MCEMP1 might be
involved in the mechanisms regulating mast cell growth and differentiation. However, there was
a long time that nearly no research on MCEMP1 was conducted after its identification until one
6
paper indicating that peripheral blood MCEMP1 gene expression might be a biomarker for
stroke prognosis (Raman et al., 2016). This research found that on average, whole-blood
MCEMP1 expression was 2.4‑fold higher in patients with stroke within 5 days of symptom onset
than in controls. This finding raised the importance of MCEMP1 in pathogenesis of disease and
as a potential therapeutic target.

It was found that there was a putative immune receptor tyrosine-based activation motif
(ITAM) in the cytoplasmic tail of MCEMP1. The molecular weight for MCEMP1 is around 20
kDa in mouse and 21 kDa in human. According to GeneCards®, there are 4 phosphorylation
sites on the intracellular domain and 3 phosphorylation sites and 1 glycosylation site on the
extracellular domain of MCEMP1 in both mice and humans.

Antibodies and Antibody Fragments as Research and Therapeutic
Tools
Antibodies are immunoglobulin (Ig) glycoproteins produced by B cells. They protect host
from infection as an important part of adaptive immunity, which processes are achieved by
specific recognition and binding of unique pathogenic molecules, which are also called antigens.
Based on this exceptional characteristic, antibodies not only become broadly exploited efficient
tools for biomedical research, but also become widely used effective drugs in therapeutics. In
1986, OKT3 – then renamed muromonab, identified by Dr. Schlossman and his collaborators
targeting a T cell-surface protein called CD3 to prevent transplant rejection in clinic by depleting
T cells, became the first therapeutic monoclonal antibody approved by FDA (Mullard, 2021).
Since then, 100 monoclonal antibody products have been approved up to 2021. Antibodies
7
become the most rapidly growing drug class. According to statistics, it may only need months to
discover an antibody that can work with a target while it always takes years for a chemical drug
seeking. Besides, an analysis of 569 antibodies from 2005 to 2014 done by Janice Reichert,
Executive Director of The Antibody Society, showed that antibody drugs were twice more likely
to succeed than small molecules during clinical trials (Mullard, 2021).

A canonical antibody is a “Y” shaped symmetrical structure with two heavy chains and
two light chains. Each heavy chain contains four domains, VH, CH1, CH2, CH3. Each light chain
contains two domains, VL and CL. VH and VL occupying the top of each chain for the “Y”
structure are variable domains that define their antigen-binding specificity. The rest domains of
an antibody are constant, with CH2 and CH3 (also called Fc) shaping the stem of the “Y”
structure. Since antibody molecules are modular and the branches of an antibody can be easily
separated from the stem through biomedical or genetic means, antigen-binding fragments (Fab)
or single chain variable fragments (scFv) (Figure 2) are frequently isolated and engineered for
their advantages as a smaller functional body compared to a full-size antibody (Nelson, 2010).
Firstly, they might be able to infiltrate some tissues or access therapeutic targets that are difficult
for full-size antibody to reach. Secondly, these smaller antibody fragments might also be
developed as tools to block a specific molecular interaction with lower chance of generating
immunogenicity. Thirdly, they could be better choices in immunohistochemistry or other
detection applications because the lack of Fc can greatly reduce non-specific binding. In
addition, they can be effectively produced in prokaryotic production systems with simpler steps,
less cost and higher yields. However, the production of these fragments still face challenges
because of the requirement for domain association (Harmsen & De Haard, 2007).
8

Interestingly, there are some exceptions to the canonical structure of antibodies found in
camelids (llama, camel, alpaca, vicugna and their relatives) and particular cartilaginous fish
(such as nurse shark and wobbegong) (Muyldermans, 2013). For such antibodies, a single
variable domain (VHH, also called nanobody) within 15 kDa can represent the entire remarkable
specificity of each antibody (Figure 2). When compared to Fab or scFv, nanobody can be easily
produced in microbial cells as a single gene with high stability and solubility, and cost-
effectively (McMahon et al., 2018).

Figure 2: A. Structure of canonical antibody in human, mouse, and most other mammals.
B. Structure of camelid antibody (llamas, camels, alpacas, and their relatives).
C. Structure of antibody fragments.

There has been much technological development for antigen-specific antibody. The
traditional approach requires the injection of antigen into laboratory or farm animals. Polyclonal
antibodies can be recovered directly from the serum of the injected animals. Monoclonal
antibodies can be obtained with a hybridoma technique by fusing antibody-secreting spleen cells
with immortal myeloma cells (Köhler & Milstein, 1975). This technique was awarded with the
1984 Nobel Prize in Physiology or Medicine. Another approach utilizes a technique by creating
phage display libraries developed by George P. Smith and Sir. Gregory Winter from mid-1980s
9
to early 1990s, which cutting-edge invention was also awarded with the 2018 Nobel Prize in
Chemistry (Almagro et al., 2019). In addition, yeast and ribosome display have also been
established (Harmsen & De Haard, 2007). The utility of display technology has become a
powerful platform for therapeutic antibody discovery. Up to the time of this review, 14 antibody
drugs produced through the display method has been approved. The success of display
technology further accelerates the development of nanobodies, as it allows flexible engineering
for enhanced functionality and fast generation in a matter of weeks (Alfaleh et al., 2020).

In this study, we used a fully synthetic yeast display nanobody library originally designed
by Dr. Andrew C. Kruse’s lab at Harvard University to identify conformationally selective
nanobodies. The library was devised using an alignment of structurally characterized nanobodies
from the Protein Data Bank (PDB) and has been trialed to successfully identify
conformationally-selective nanobodies for two human GPCRs with cell-based selection scheme
enabled by magnetic cell sorting (MACS) and fluorescence activated cell sorting (FACS)
(McMahon et al., 2018).

Summary of Chapter 1
Mast cells and their activation can be detrimental to human health. KIT is one of the most
important receptors for the proliferation and activation of mast cells. Reducing mast cell numbers
and preventing mast cell differentiation by inhibiting KIT receptor has been proved to be a good
approach to treat mast cell-driven diseases as evinced by currently approved drugs. A
preliminary study in Dr. Jung’s lab showed that MCEMP1 positively regulates KIT receptor
activation in an ITAM-dependent manner in vitro. Therefore, a nanobody inhibiting MCEMP1
can be a useful tool to investigate the mechanism underlying MCEMP1-mediated regulation of
10
KIT receptor on the surface of mast cells as well as a good drug candidate in treating mast cell-
driven inflammatory lung diseases. In this study, HEK 293T cells were utilized to produce the
ectodomain of MCEMP1 (MCECTO) of mouse and the antigen-specific nanobodies were
screened with a synthetic yeast display nanobody library from Dr. Kruse’s lab by MACS and
FACS.

11
Chapter 2: Large-Scale Protein Production and their Purification
Introduction
For nanobody selection, milligrams of target proteins need to be obtained by molecular
cloning. There are many options of expression systems for protein production, such as E. coli,
yeast, mammalian cells, insect cells and cell-free systems. HEK 293 cells, a cell line generated
by transfection of normal human embryonic kidney (HEK) cells with sheared DNA fragments of
human adenovirus type 5 (Ad5) (Thomas & Smart, 2005), have become the most ideal
mammalian cell line for lab-scale protein production for their ease of culture and high efficiency
of transfection (Nettleship et al., 2015). Here we chose HEK 293T, a useful variant of HEK 293
cells expressing the SV40 large T antigen with increased transfected copy numbers of plasmid
containing the SV40 origin of replication and thus higher levels of transient expression
(Nettleship et al., 2015), as an expression system to produce secreted protein MCECTO.
Purifications were performed by immunoprecipitation method based on FLAG epitope tagged
MCECTO.

MCEMP1 Ectodomain (MCECTO) as an Antigen
MCEMP1 is a transmembrane protein expressed on the surface of mast cells. The
ectodomain of a transmembrane protein extends into the extracellular space and is usually
thought to contact with the cell-surface signal and to induce signal transduction (Ryu et al.,
2019). This study aimed to discover a nanobody as a research and therapeutic tool. Our strategy
was to identify a nanobody that binds to the extracellular domain of MCEMP1 and inhibits its
12
function. To reduce non-specific binding, I chose MCECTO solely as the antigen rather than the
whole MCEMP1 protein for nanobody selection.

MCECTO Cloning and Transient Gene Expression of 293T Cells
There are several systems to produce recombinant protein in a lab setting. Prokaryotic
systems like E. coli are not capable to fulfill post-translational modifications including
glycosylation that is related to protein solubility and /or biological activity required by
eukaryotic proteins (Correa & Oppezzo, 2015). Insect cells perform similar post-translational
modifications present in mammalian proteins, but their metabolic pathways of glycosylation are
distinct from mammalian cells (Ferrer-Miralles et al., 2015). Yeast expression systems are
expected to be a main potential source of producing human glycoproteins, however none of the
commercialized therapeutic proteins produced in S. cerevisiae to date are glycosylated (Ferrer-
Miralles et al., 2015).

MCEMP1 is a mammalian protein with a glycosylation site on its ectodomain.
Considering the post-translational modification that troubled expression systems like bacteria,
insects, or yeasts of producing mammalian proteins, a mammalian cell line became my first
choice. In addition, I chose an inexpensive cationic polymer polyethylenimine (PEI) as the
DNA-condensing reagent, which is routinely used for production of secreted and cell surface
glycoproteins due to its feasibility for a large-scale transient transfection of HEK 293 cells
(Aricescu & Owens, 2013). I performed transient gene expression in HEK 293T cells and
produced milligrams of recombinant proteins. I used pFUSEN-mG2AFc plasmid as a vector that
contains IL-2 signal peptide (Figure 3A), thus mouse protein MCECTO could be directly
13
secreted out of cells into the cell culture media, which made it easy to harvest recombinant
protein and to conduct protein purification. I also used pFUSEN-hG1Fc plasmid for the cloning
of human MCECTO (Figure 3B) and saved the recombinant plasmids for future study.

Figure 3: A. pFUSEN-mG2AFc plasmid backbones used for mouse MCECTO cloning.
B. pFUSEN-hG1Fc plasmid backbones used for human MCECTO cloning.

Design of Recombinant Plasmid and MCECTO Purification
In this study, three different recombinant plasmids for mouse MCECTO were designed
and cloned. For the first two constructs, Fc domain in the backbone of pFUSEN-mG2AFc
plasmid was removed and a FLAG-tagged MCECTO was inserted (Figure 4: Construct 1&2).
FLAG tag (DYK-DDDDK) was inserted either at N terminal of the mouse MCECTO or at its C
terminal. The third construct kept the Fc domain in the pFUSEN-mG2AFc plasmid and included
a PreScission protease cleavage site (Leu-Phe-Gln/Gly-Pro) between Fc domain and the mouse
MCECTO, and FLAG tag was inserted at the C terminal of mouse MCECTO (Figure 4:
Construct 3). For production of human MCECTO protein, gene was inserted into pFUSEN-
hG1Fc plasmid with the PreScission protease cleavage site (Figure 4: Construct 3).
A.
B.
14

Figure 4: Three different DNA constructs that were designed to be cloned in HEK 293T cells.

The FLAG-tagged mouse MCECTO proteins (construct 1&2) were immunoprecipitated
with ANTI-FLAG M2 affinity gel and eluted with FLAG peptide (Figure 5: Strategy1). Fc-
conjugated mouse MCECTO fusion protein was immunoprecipitated with Pierce Protein A/G
Agarose (usually called A/G beads), which bound to the Fc domain of Fc-MCECTO
recombinant protein, followed by PreScission Protease cleavage that allows MCECTO releasing
from Fc-A/G beads. The PreScission Protease was removed by Pierce GST Agarose (Figure 5:
Strategy 2).

Figure 5: A. Strategy 1 for MCECTO purification without Fc (construct 1&2).
B. Strategy 2 for MCECTO purification with Fc (construct3).

Construct1:
Construct2:
Construct3:
A.
Strategy 1 Strategy 2
B.
15
Optimization of Cell Culture Routine and Protein Purification
The cell culture system and purification method for production of mouse MCECTO
protein were optimized for protein yield. An initial routine for HEK 293T cell culture was as
follows: Firstly, PEI transfection was performed when cells were 80% confluent in a plate.
Secondly, after 24 hours of PEI transfection, cell culture media were changed from complete
DMEM (DMEM + 10% FBS + 1% PS) to FreeStyle™ 293 Expression Media. Thirdly, after 72
hours of PEI transfection, media containing mouse MCECTO protein was harvested and
subjected to protein purification (Routine 1). During optimization, the cell culture routine was
improved in following ways: using next generation high cell density-supportive Expi293™
Expression Medium as a substitute for FreeStyle™ 293 Expression Media (Routine 2); adding
0.3M valproic acid to Expi293™ Expression Medium based on routine 2 (Routine 3); improving
protocol for transfection (details in the Methods Section) by changing media 8 hours rather than
24 hours after transfection based on routine 3 condition, and concentrating the harvested media
by tangential flow filtration (TFF) before purification (Routine 4).

Three rounds protein purification of mouse FLAG-tagged MCECTO (Figure 5: Strategy
1) were performed. For the first round, 18 plates of 100 mm Corning® Tissue Culture Dishes of
70% confluent HEK 293T cells were divided into two groups with 9 plates each group. One
group of cells were transfected with recombinant plasmid with N-terminal FLAG-tagged
MCECTO (Figure 4: Construct 1) and the other group of cells were transfected with C-terminal
FLAG-tagged MCECTO (Figure 4: Construct 2). After PEI transfection, complete DMEM
media were not changed into FreeStyle™ 293 Expression Media and purification was not
performed for this round (Figure 6A: 1st). Whole cell lysates and media of each group was
16
immunoblotted (Figure 6B). In result, the C-terminal FLAG-tagged MCECTO showed higher
levels of protein expression than the N-terminal FLAG-tagged MCECTO.

For the second round of purification, 12 plates of 100 mm Corning® Tissue Culture
Dishes of HEK 293T cells were transfected with recombinant plasmid Construct 2 (Figure 6A:
2nd). Transfection protocol followed Routine 1. Cell culture media containing MCECTO were
harvested and subjected to protein purification strategy 1. The final volume of eluted protein was
200 µL. The concentration of purified protein was estimated by performing Coomassie-stained
SDS-PAGE with standard gradient concentration of BSA protein (Figure 6C). The estimated
concentration of MCECTO was 25 µg/mL. In all, the estimated yield of the 2nd round of
transfection was 5 µg.

For the third round of purification, 50 plates of 150 mm Corning® Tissue Culture Dishes
of HEK 293T cells were transfected with recombinant plasmid Construct 2 (Figure 6A: 3rd).
The transfection protocol followed Routine 1. Cell culture media containing MCECTO were
harvested and concentrated into 26 ml with Amicon® Ultra-15 centrifugal filter 3K devices and
were further purified following purification strategy 1. The final volume of eluted protein was
200 µL. The total of 10 ug MCECTO protein was obtained by an estimation with Coomassie-
stained gel. By looking into the details of the third-round transfection for MCECTO, I found that
the approximate growth area and the average cell yield of different sizes of Corning® dishes
could not be simply calculated and compared by their radiuses. By doing so, only twice amounts
of DNA and PEI were added to each 150 mm tissue culture dish compared to each 100 mm
tissue culture dish. However, the approximate growth area and the average cell yield of one 150
17
mm dish are three times of one 100mm dish according to a guide released by Corning®. This
might be a main reason that led to the reduction of the yield of MCECTO per cell and was only
corrected in routine 4. The yield of protein concentrated with Amicon® Ultra-15 centrifugal
filter 3K devices was poor when the volume of harvested media was too large for the capacity of
the device, which could be another reason for the protein loss.

Figure 6: A. Cell-culture for three rounds of transfection of FLAG-tagged MCECTO plasmid.
B. Western blot for FLAG-tagged MCECTO in cell lysate and media after first-round transfection.
C. Coomassie-stained SDS-PAGE for purified MCECTO and gradient concentration of BSA proteins.

(Routine 1)
B. C.
A.
5ug
10ug
Different
Contrast ratio
18
Next, I performed protein purification with strategy 2 and construct 3, which the
recombinant protein contains Fc domain that was cleaved off and removed during purification
process.

Figure 7: A. Cell-culture routines for seven rounds of transfection of Fc-MCECTO recombinant plasmid.

Seven rounds of transient expression of MCECTO with Fc and their purification (Figure
5: Strategy 2) were performed. For the first round, 19 plates of 100 mm Corning® Tissue
Culture Dishes of HEK 293T cells were transfected (Figure 7: 1st). The A/G beads bound
MCECTO proteins were first incubated with 8 µL PreScission Protease for 4 hours (200 µL
elution) and the remaining A/G beads were further incubated with additional 8 µL PreScission
Protease for overnight (200 µL elution). The total of eluted 400 µL MCECTO protein were
incubated with GST Agarose beads to remove PreScission Protease. I performed Coomassie-
stained SDS-PAGE and western blot with the purified MCECTO and the leftover of Pierce
Protein A/G Agarose (Figure 8). Fair amounts of MCECTO proteins were remained in the A/G
Agarose leftover and Pierce GST Agarose leftover. Thus, I decided to include additional elution
steps to increase the protein yield. BCA Protein Assay was performed to determine the final
19
concentration of purified MCECTO protein. I found that the solvent of cleavage buffer contained
DTT, a reducing substance, interfered with the BCA Protein assay. Thus, I included dialysis
procedure to change the cleavage buffer into PBS before BCA Protein Assay for concentration
estimation.

Figure 8: Coomassie-stained SDS-PAGE and western blot for the 1st purification of Strategy 2.

I scaled up for the second round of purification. 50 plates of 150 mm Corning® Tissue
Culture Dishes of HEK 293T cells were transfected (Figure 7: 2nd). The transfection protocol
followed Routine 1. I added one additional elution step with another 200 µL cleavage buffer. The
final volume of purified proteins was 600 µL. The concentration of the purified MCECTO
protein was 100 µg/mL estimated by SDS-PAGE and Coomassie brilliant blue staining with
standard gradient concentration of BSA protein (Figure 9A). The yield of MCECTO was 60 µg.
Subsequently, I washed both Pierce Protein A/G Agarose and Pierce GST Agarose three times
separately with 100 µL, 200 µL and 200 µL cleavage buffer to determine whether additional
washing/elution steps were needed. With the Coomassie and immunoblotting, I found that
additional three times of washing steps can elute most of the purified MCECTO (Figure 9B).
20
Thus, 500 µL purified MCECTO protein after three times of additional elution were combined
with the initial 600 µL purified MCECTO protein. Finally, the total volume of purified
MCECTO protein of this round was 1100 µL. I also combined them with 400 µL MCECTO
protein obtained from the first-round purification, yielding 1.5 mL of total purified proteins.
After dialysis, BCA Protein Assay estimated protein concentration of 100 µg/mL (Figure 9C).
As a result, the yield of the two rounds of purification was 150 µg.

Figure 9: A. Coomassie-stained SDS-PAGE for purified MCECTO obtained in the 2
nd
purification of Strategy 2 and
gradient concentration of BSA proteins.
B. Western blot for additional washing/elution of A/G agarose and GST beads.
C. BCA Protein Assay to estimate the concentration of Strategy2_1
st
+2
nd
purified MCECTO.

A. B.
C.
21
For the third and fourth round, 50 plates of 100 mm Corning® Tissue Culture Dishes of
HEK 293T cells were transfected separately (Figure 7: 3rd & 4th). The transfection protocol
followed Routine 1. Based on the second-round of purification, I washed both Pierce Protein
A/G Agarose and Pierce GST Agarose with 200 µL cleavage buffer three times. The total of 1.6
mL purified MCECTO were obtained for both the third and the fourth round. The final
concentration of the third-round purified protein was around 125 µg/mL as estimated by
performing Coomassie-stained SDS-PAGE with standard gradient concentration of BSA (Figure
10A). The yield of third-round transfection was 200 µg. The yield of the fourth round of
purification was similar to the third round of purification (Figure 10B).

Figure 10: A. Coomassie-stained SDS-PAGE for purified MCECTO obtained in the third purification of Strategy 2
and gradient concentration of BSA proteins.
B. Coomassie-stained SDS-PAGE for purified MCECTO obtained in the fourth purification of Strategy 2
and gradient concentration of BSA proteins.

For the fifth round of purification, 55 plates of 150 mm Corning® Tissue Culture Dishes
of HEK 293T cells were transfected (Figure 7: 5th). The transfection protocol followed Routine
2. The Expi293™ Expression Medium was used as a substitute for FreeStyle™ 293 Expression
A. B.
22
Media. The first 200 µL purified MCECTO was accidentally lost. The concentration of proteins
was about 50~75 µg/mL and the yield were about 125 µg (Figure 11).

Figure 11: Coomassie-stained SDS-PAGE for purified MCECTO obtained in the fifth purification of Strategy 2 and
gradient concentration of BSA proteins.

For the sixth round of purification, 60 plates of 150 mm Corning® Tissue Culture Dishes
of HEK 293T cells were transfected (Figure 7: 6th). The transfection protocol followed Routine
3, routine 2 plus 0.3M valproic acid included in PEI transfection. After purification, I obtained 2
mL of MCECTO protein. The purity and concentration for this round was estimated by
Coomassie-stained SDS-PAGE and BCA Protein Assay after dialysis. The concentration of
protein was 183 µg/mL, and the yield of protein was 366 µg for the 6th round of purification
(Figure 12).

23
Figure 12: A. Coomassie-stained SDS-PAGE for purified MCECTO obtained in the sixth purification of Strategy 2
and gradient concentration of BSA proteins.
B. Color response curves for BSA in the BCA Protein Assay to estimate the concentration of
Strategy2_6th purified MCECTO.

For the seventh round of purification, 60 plates of 150mm Corning® Tissue Culture
Dishes of HEK 293T cells were transfected (Figure 7: 7th). The transfection protocol followed
Routine 4. I modified procedure based on Routine 3. I changed media 8 hours rather than 24
hours after PEI transfection and harvested media was concentrated with TFF to 30 mL before
purification. The total of 2.9 mL of MCECTO protein was purified and subjected to dialysis.
BCA assay showed protein concentration of 58 µg/mL (Figure 13). The yield was about 1.7 mg.

Figure 13: Color response curves for BSA in the BCA Protein Assay to estimate the concentration of Strategy 2_7th
purified MCECTO.

In summary, after several rounds of MCECTO protein purification ultimately yielded
2.756mg antigen to perform nanobody screening with a recently developed synthetic nanobody
library displayed in yeast.

24
Chapter 3: Identification of Nanobodies that Bind to Protein
MCECTO
Introduction
Different from conventional antibodies, camelid antibodies are composed solely of heavy
chains, a single variable domain (nanobody) of which, contains the entire antigen-binding
surface. Nanobodies can be readily expressed as the product of a single gene in bacteria with
retained antigen specificity. This characteristic allows the use of library display technologies for
in vitro screening of nanobody, which bypasses time-consuming and costly animal
immunization. There were commercial providers combining synthetic library with phage-
display, which can isolate high-affinity binders, but it was still difficult to gain functional clones.
A yeast-display library developed by Dr. Kruse’s lab aimed to solve this problem and
successfully isolated conformationally selective nanobodies for two human GPCRs (McMahon
et al., 2018). Based on a consensus framework derived from llama gene IGHV1S1 –IGHV1S1S5,
the fully synthetic yeast-display DNA library of nanobodies was constructed by an alignment of
structurally characterized nanobody clones from the Protein Data Bank (PDB). To achieve
diversity of the nanobody library, randomizations of amino acids were introduced to the
complementarity-determining loops (CDRs) that comprise the highly variable antigen-binding
interface of the VHH and residues adjacent to CDRs. These randomizations of amino acids in
CDRs emulating their frequencies in all nanobodies in PDB were position-specifically analyzed.
Besides, a long flexible stalk was designed to covalently tether the nanobody to the yeast cell
wall with an HA epitope tag at the C terminal of the nanobody. The linearized engineered
surface display plasmid pYDS649 was transformed into Saccharomyces cerevisiae protease-
25
deficient strain BJ5465 and generated a yield of 5x10
8
transformants, which contains at least
1x10
8
unique full-length nanobodies that can be expressed and displayed on the yeast surface. I
used this yeast-display library for screening MCECTO-specific nanobodies with high-affinity
that can promote or inhibit the function of MCECMP1.

Yeast-based Nanobody Selection by Magnetic Cell Sorting (MACS)
I sought to identify novel nanobodies targeting MCECTO protein by yeast cell-based
selection process that was composed of two rounds of magnetic cell sorting (MACS) followed by
two rounds of fluorescence activated cell sorting (FACS). MACS is a method developed by
Miltenyi Biotec for separation of cells with different surface antigens by using magnetic
nanoparticles (usually called magnetic beads) coated with antibodies against a particular surface
antigen. The whole set of devices for yeast selection contained a QuadroMACS™ Separator, LD
columns for negative selection (depletion of yeast with reactivity to magnetic beads or secondary
antibodies) and LS columns for positive selection (isolates clones that bind to MCECTO
antigen). The negative selection is important to ensure that binders specifically recognize the
antigen, thus the depletion step was conducted before every selection step. Here, I used Anti-
Mouse IgG1 MicroBeads for magnetic labeling and subsequent cell separation and used
Monoclonal ANTI-FLAG® M2 antibody produced in mouse to identify yeast expressing
antibodies against purified FLAG-tagged MCECTO. In brief, the yeast library was incubated
with FLAG-tagged MCECTO antigen that were pre-incubated with Monoclonal ANTI-FLAG®
M2 antibody, followed by staining with Anti-Mouse IgG1 MicroBeads. Labeled cells were then
isolated by magnet-based separation and amplified in standard yeast culture medium. Before
each selection, yeast was induced with galactose media and nanobody expression was checked
26
with a quick analytical flow cytometry assay (details in the Methods Section). The maximum
number of expressing cells from the naïve library is ~25%. The typical number of expressing
cells is ~15-20%. According to the protocol of the nanobody library, if expression is on the low
end (~8-12%), increasing the number of cells used for nanobody selection is considered to
compensate. I obtained a very low expression rate of nanobody (3.63 %) (Figure 14A). After the
1
st
MACS selection, 0.3% yeast cells were recovered. The recovered yeast cells were frozen to
be induced before the next selection. On the day of the 2
nd
MACS selection, the nanobody
expression rate was 6.59% when it was tested strictly following the protocol from Dr. Kruse’s
lab and was 41.1% when it was test less stringent (Figure 14B). After the 2
nd
MACS selection,
0.58% yeast cells were recovered.

Figure 14: A. nanobody expression level before 1
st
MACS on flow cytometer with unstained sample to set gates.
B. nanobody expression level before 2
nd
MACS on flow cytometer with unstained sample to set gates
in two different ways.

A. B.
27
Yeast-based Nanobody Selection by Fluorescence Activated Cell
Sorting (FACS)
To enrich for the desired MCECTO-specific nanobodies, I subsequently performed two
rounds of FACS selection. The yeast library was incubated with purified FLAG-tagged
MCECTO and APC anti-DYKDDDDK (FLAG) Tag Antibody. The yeast cells labelled with
APC anti-DYKDDDDK were sorted with BD FACSAriaTM III flow cytometer. After the first
round of FACS sorting, 0.06% positive cells were recovered (Figure 15A). After the second
round of FACS sorting, 0.74% positive cells were recovered (Figure 15B).

Figure 15: A. cells sorted after 1
st
FACS.
B. cells sorted after 2
nd
FACS.

Binding Test for Sorted Clones and MCECTO
Adenine-deficient pink yeast appeared after the four rounds of selection. I categorized
these individual clones into adenine sufficient group (normal white cells) and adenine deficient
cells (abnormal pink cells) and randomly picked out 5 clones from each group to check their
A. B.
28
capacity of binding to MCECTO with the quick analytical flow cytometry assay by Life Tech
Attune NxT flow cytometer (Figure 16A). In brief, each clone picked out were amplified and
incubated with purified FLAG-tagged MCECTO and PE anti-DYKDDDDK (FLAG) Tag
Antibody on a 96-well U-bottom plate. The adenine-sufficient group showed higher binding
capacity (1.14%, 12.4%, 1.33%, 2.07% and 3.79%) compared to MCECTO than the adenine-
deficient group (0.27%, 0.18%, 0.19%, 0.12% and 0.17%) (Figure 16B).

Figure 16: A. comparison of adenine-deficient yeast and adenine-sufficient yeast after the 4
th
selection by FACS.
B. The result of yeast and MCECTO binding affinity for two groups and the example from the 2
nd

colonies of both groups.

To obtain more colonies of yeast that had high binding affinity with MCECTO, seventy
more adenine-sufficient single clones were picked out to check binding capability to MCECTO
(Figure 17A). The result showed that 11 clones had >9% MCECTO-binding capability
(categorized as high binding) and 3 clones had >5% MCECTO-binding capability (categorized
as moderate binding) (Figure 17B). These 14 clones were chosen for further study.
A. B.
29

Figure 17: A. Picking out another 70 different adenine-sufficient yeast colonies after the 4
th
selection by FACS.
B. The result of yeast and MCECTO binding affinity for another 70 different adenine-sufficient
yeast colonies.

Nanobody Sequencing and Preparation for Nanobody Purification
in E. coli
I isolated yeast plasmid DNA with Phenol, Chloroform, and Isoamyl Alcohol (PCI)
method and electroporated these plasmid DNA into electrocompetent Top10 E. coli cells to
obtain enough amounts of DNA for sequencing. The sequencing results showed 12 unique
sequences out of 14 clones. Among them, *3 and *8 clones, #9 and #12 clones showed the same
sequence (Figure 18). These four clones all belonged to the high MCECTO-binding capacity
group. Only one clone had higher binding capacity than these four clones. The top three high
binding capacity clones (star candidates) were chosen for further nanobody purification.
A. B.
30

Figure 18: A. The MCECTO binding affinity of all 14 yeast clones with their nanobodies that were sequenced.
B. The sequences of 3 nanobodies with their yeast cells among the highest binding affinity with
MCECTO.

Extracted yeast plasmids were used as templates to amplify nanobody DNA sequences by
polymerase chain reaction (PCR). Amplified nanobody DNA sequences were then cloned into
plasmid pET-26b by Gibson cloning and transformed into TOP10 for sequencing to confirm the
success of cloning. pET-26b containing nanobody genes were ready for nanobody purification
with BL21 cells.

A.
B.
31
Chapter 4: Discussion
Before Ramen’s paper showed that peripheral blood MCEMP1 gene expression might be
a biomarker for stroke prognosis in 2016, nearly no research about the biological function of
MCEMP1 was published since its identification in 2005. In Dr. Choi’s unpublished study,
MCEMP1 contains a functional ITAM that can transduce signals to induce the activation of
MAPK downstream kinase cascade, activation of NF-κB pathway, intracellular calcium
mobilization, chemokine production and degranulation. It was also found that MCEMP1 can
enhance KIT phosphorylation and is essential for optimal mast cell expansion.

Herein, I aim to identify a MCEMP1-specific nanobody that can be used not only as a
research tool to study a biological function of MCEMP1, but also as a potential therapeutic
agent. I optimized cell culture and transfection system for HEK 293T cells and purification
method for MCECTO. I obtained 1.7mg of protein MCECTO by transfecting 60 plates of 150
mm Corning® Tissue Culture Dishes of HEK 293T cells. In total, I obtained 2.756mg of the
protein MCECTO after ten rounds of transfection and purification. I sorted out 0.74% of positive
yeast cells targeting antigen MCECTO after two rounds of MACS and two rounds of FACS
screening. One limitation of this study is that nanobody expression rate of our induced yeast
library was much lower than it was indicated in the protocol from Dr. Kruse’s lab when it was
tested strictly. This could be a main reason that I sorted out very low rates of positive cells after
each round of selection. I confirmed that adenine-deficient yeast cells showed minimal nanobody
expression and should be eliminated during screening. I identified 11 yeast colonies with >9%
positive rates and 3 colonies with >5% positive rates of MCECTO-binding affinity out of 75
different adenine-sufficient yeast colonies tested with a quick analytical flow cytometry assay. I
32
sequenced nanobodies in these 14 yeast colonies and further analyzed their binding affinity with
MCECTO. Among 14 nanobodies, there were two nanobodies that appeared twice in sequencing
results and showed high binding affinity to MCECTO. In addition, there was one nanobody
showed higher binding affinity than the two nanobodies mentioned above. In summary, these
three nanobodies were selected to further characterize for mouse MCEMP1-specific inhibitory
nanobodies.

Future study will identify an inhibitory nanobody that blocks MCEMP1-induced
downstream signaling. This nanobody will be useful to investigate a function of MCEMP1 as an
adaptor for SCF-KIT-mediated mast cell proliferation. A therapeutic capacity of MCEMP1
nanobody will be further characterized using a mouse asthma model to see if it can relieve
asthma conditions.

33
Chapter 5: Materials and Methods
Cells
HEK 293T cells were purchased from ATCC (CRL-3216) and were cultured in complete
DMEM (cDMEM), which contained Dulbecco's Modified Eagle Medium (DMEM) (Thermo
Fisher Scientific, Waltham, MA) + 10% Fetal Bovine Serum (FBS) (Clontech, San Jose, CA)
+1% Pen Strep (P/S) (Thermo Fisher Scientific, Waltham, MA) when grown as adherent cells in
100 mm / 150 mm Corning® Tissue Culture Dishes (Corning, Glendale, AZ) in a 37°C incubator
with 5% CO2.

Transfection
For transfection of adherent HEK 293T, cells were seeded in 100 mm / 150 mm
Corning® Tissue Culture Dishes to be 70% confluent the following day. On the day of
transfection for each 100 mm dish, 10 ug DNA was mixed with 40 µL polyethylenimine (PEI) at
1 mg/mL in 300 µL serum free DMEM, incubated for 10 minutes at room temperature and then
was mixed well with 500 µL cDMEM and was added dropwise to cells. The amount of
transfection reagents used for a 150 mm culture dish in routine 1, 2 and 3 was double of a 100
mm culture dish, which was supposed to be three times and was corrected in routine 4. For
Routine 1, media was changed to 6 mL / 12 mL FreeStyle™ 293 Expression Media per dish 24
hours after transfection. For Routine 2, media was changed to 6 mL / 12mL Expi293™
Expression Media per dish 24 hours after transfection. For Routine 3, media was changed to 6
mL / 12 mL Expi293™ Expression Media containing 0.3M valproic acid per dish 24 hours after
PEI transfection. For Routine 4, the process of transfection was optimized and performed in the
34
morning for each 150 mm dish as follows, first, changing the media to 16 mL DMEM + 10%
FBS, secondly, incubating 30 µg DNA with 1mL Opti-MEM™ I Reduced Serum Medium
(Thermo Fisher Scientific, Waltham, MA) for 5 minutes, thirdly, adding 330 µL PEI at 1mg/mL
to the incubated DNA-media mixture and incubated for another 5 minutes and fourthly, adding
the PEI mixture dropwise to cells. After 4~8 hours incubation of the transfected cells at 37°C,
media was changed to 12 mL Expi293™ Expression Media containing 0.3M valproic acid per
dish. For each round of transfection, one dish of HEK 293T cells was transfected with the DNA
of green fluorescent protein (GFP) and was observed under a microscope for the assessment of
the success of transfection.

MCECTO Purification
Expression media of adherent HEK 293T cells containing secreted MCECTO was
harvested 72 hours after PEI transfection and was collected in 50 mL CELLSTAR® Centrifuge
Tubes (Greiner Bio-One, Monroe, NC). For the total 3 rounds of transfection with Strategy 1,
two rounds of harvested MCECTO were purified (round 2 and round 3). For round 2, first,
harvested media was incubated with 100 µL Glutathione Sepharose® 4B (GE Healthcare,
Chicago, IL) in each tube on a rotator in 4°C cold room for 1 hour. Secondly, media was
centrifuged at 1,000g and 4°C for 5 minutes to remove Glutathione Sepharose® 4B and then
incubated with 80 µL / 100 mm dish ANTI-FLAG M2 affinity gel (Sigma-Aldrich, St. Louis,
MO) on a rotator in 4°C cold room for 1 hour. Thirdly, ANTI-FLAG M2 affinity gel bound with
FLAG-tagged MCECTO was spun down at 1,000g and 4°C for 5 minutes and combined in a 1.5
mL Copolymer Microcentrifuge Tube (USA Scientific, Ocala, FL). Fourthly, the combined
sediments were washed with 1mL phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis,
35
MO) 5 times by centrifugation at 1,000g and 4°C for 5 minutes. Fifthly, the washed sediments
were incubated with 100 µL PBS containing FLAG® Peptide (MilliporeSigma, Burlington, MA)
at a concentration of 150 µg/mL on a rotator in 4°C cold room for 1 hour. Sixthly, purified
MCECTO were obtained in 100ul supernatant by precipitating the ANTI-FLAG M2 affinity gel
at 1,000g and 4°C for 5 minutes. Seventhly, the last two steps were repeated to obtain another
100 µL supernatant containing purified MCECTO. Both Glutathione Sepharose® 4B and ANTI-
FLAG M2 affinity gel were washed with PBS 3 times by centrifugation at 1,000g and 4°C for 3
minutes before use. Supernatant after spinning down ANTI-FLAG M2 affinity gel bound with
FLAG-tagged MCECTO and precipitated ANTI-FLAG M2 affinity gel after removing the last
100ul supernatant containing purified MCECTO were saved for SDS-PAGE. For round 3, media
containing FLAG-tagged MCECTO were first concentrated with Amicon® Ultra-15 centrifugal
filter 3K devices (Millipore-Sigma, Burlington, MA) into 30mL collected in two 15 mL
CELLSTAR® Centrifuge Tubes (Greiner Bio-One, Monroe, NC) before purification. The same
purification process was performed as round 2 and the amounts of reagents used were changed
accordingly.

Seven rounds of purification were performed with Strategy 2. For round 1 to round 4,
first, harvested media was incubated with 100 µL Glutathione Sepharose® 4B in each tube on a
rotator in 4°C cold room for 1 hour. Secondly, media was centrifuged at 1,000g and 4°C for 5
minutes to remove Glutathione Sepharose® 4B and then incubated with 5 µL/100 mm dish
Pierce Protein A/G Agarose (Thermo Fisher Scientific, Waltham, MA) on a rotator in 4°C cold
room for 2~3 hours and precipitated Pierce Protein A/G Agarose bound with MCECTO by
centrifugation at 1000g and 4°C for 5 minutes. Thirdly, Pierce Protein A/G Agarose bound with
36
FLAG-tagged MCECTO was spun down at 1,000g and 4°C for 5 minutes and combined in a 1.5
mL microcentrifuge tube. Fourthly, the combined Pierce Protein A/G Agarose was washed with
1 mL PBS 4 times and the fifth were washed with 1 mL cleavage buffer by centrifugation at
1,000g and 4°C for 5 minutes. The cleavage buffer was made of autoclaved water containing 50
mM Tris-HCl, pH 7.0 (at 25 °C), 150 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT). It
was chilled to 5 °C prior to use and DTT was only added immediately before use. Fifthly, the
washed Pierce Protein A/G Agarose bound with FLAG-tagged MCECTO was incubated with
200 µL cleavage buffer containing 8 µL PreScission Protease (Sigma-Aldrich, St. Louis, MO) on
a rotator in 4°C cold room for 2~4 hours. Sixthly, 200 µL supernatant containing the protein
MCECTO and the protease were obtained by centrifugation at 1,000g and 4°C for 3 minutes.
Seventhly, the sediments from last step were saved and was incubated with 200 µL cleavage
buffer containing 8 µL PreScission Protease overnight to obtain another 200uL supernatant
containing the protein MCECTO and the protease. Eighthly, the obtained supernatants were
combined in a 1.5 mL microcentrifuge tube and incubated with Pierce GST Agarose (Thermo
Fisher Scientific, Waltham, MA) on a rotator in 4°C cold room for 1 hour. Ninthly, purified
MCECTO was obtained in 400 µL supernatant by precipitating Pierce GST Agarose bound with
the protease at 1,000g and 4°C for 3 minutes. Glutathione Sepharose® 4B, Pierce Protein A/G
Agarose and Pierce GST Agarose were washed with PBS 3 times by centrifugation at 1,000g and
4°C for 3 minutes before use. Supernatant after spinning down Pierce Protein A/G Agarose
bound with FLAG-tagged MCECTO and precipitated Pierce Protein A/G Agarose after
removing supernatant containing MCECTO were saved for SDS-PAGE. For round 5 and round
6, Pierce Protein A/G Agarose was cleaved in 400 µL cleavage buffer with 16 µL PreScission
protease for 4 hours and for overnight. For round 7, media was concentrated to 30 µL by
37
Tangential Flow Filtration (TFF) before purification. Pierce Protein A/G Agarose was only
cleaved once overnight in 600 µL cleavage buffer with 48 µL PreScission protease.

Based on the result of round 1, Pierce Protein A/G Agarose and Pierce GST Agarose
were washed with cleavage buffer after obtaining supernatant containing MCECTO in the
following purification processes for higher yield. For round 2, initially, A/G Agarose was only
washed once after obtaining 2
nd
supernatant containing MCECTO by centrifugation at 1,000g
and 4°C for 3 minutes and prepared for SDS-PAGE. According to Coomassie and western blot,
more washing steps were needed. Therefore, both the Pierce Protein A/G Agarose leftover and
the Pierce GST Agarose leftover was washed 3 times with 100 µL, 200 µL, 200 µL cleavage
buffer and redissolved in another 200 µL cleavage buffer. Supernatant obtained after each
washing were collected and prepared for SDS-PAGE. Redissolved Agaroses were also prepared
for SDS-PAGE. For round 3 to round 6, both Pierce Protein A/G Agarose and Pierce GST
Agarose were washed 3 times with 200 µL cleavage buffer and collected more 1200 µL purified
MCECTO by washing steps. For round 7, the A/G Agarose was washed 3 times with 400 µL,
800 µL, 200 µL cleavage buffer and the Pierce GST Agarose was washed with 300 µL cleavage
buffer 3 times and collected more 2300 µL purified MCECTO by washing steps. Purified
MCECTO were stored at -80°C.

SDS-PAGE, Coomassie and Western Blot
The results of production and purification of MCECTO were assessed by SDS-PAGE
with Coomassie staining and western blot. All protein samples were mixed with 6xSDS or
2xSDS buffer and boiled at 95°C for 5 minutes to negative charge and denature proteins and then
38
ran on 18% gels for 80 minutes at 120v using a Bio-Rad mini Protean system. For Coomassie
blue staining, the gels were incubated with InstantBlue™ (VWR, Radnor, PA) while gently
rocking overnight. For western blot, proteins on gels were transferred to PVDF membranes (Bio-
Rad Laboratories, Hercules, CA) by semi-dry transfer at 25v for 45 minutes with Trans-Blot
Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with 1g
dry milk dissolved in 20 mL Tris Buffered Saline, with Tween® 20, pH 8.0 (TBST) (Sigma-
Aldrich, St. Louis, MO) while gently rocking for 1 hour and washed with TBST once.
Membranes were then incubated with primary antibodies Monoclonal ANTI-FLAG® M2
antibody produced in mouse (Sigma-Aldrich, St. Louis, MO) and milk-TBST solution at 1:1000
while gently rocking for 2 hours. After washing 3 times with TBST by gently rocking for 5
minutes, membranes were incubated with HRP-conjugated anti-mouse-IgG secondary antibodies
with milk-TBST solution at 1:5000 while gently rocking for 1 hour. Membranes were then
washed three times with TBST. Membranes were left in TBST from last wash and stained with
ECL reagent. Protein bands were developed with ECL reagent and imaged on a Bio-Rad
ChemiDoc imaging system.

Dialysis and BCA Assay
Purified MCECTO were dialyzed with Slide-A-Lyzer™ Dialysis Cassettes, 3.5K
MWCO, 3 mL (Thermo Fisher Scientific, Waltham, MA) by PBS. Concentrations of dialyzed
MCECTO were assessed with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific,
Waltham, MA) by following microplate procedure and preparing a set of albumin standard at 0,
25, 125, 250, 500, 750, 1000, 1500, 2000 µg/mL. Two replicates were performed for each assay.

39
Yeast Nanobody Selection
The naïve yeast library (NbLib) at a density of 10
10
cells/ml were thawed and 250ul were
induced in 250ml of -Trp + galactose media followed by shaking for 72 hours, 25 °C, 220 rpm.
Cell densities were calculated by measuring their optical densities with a spectrophotometer at
OD600. Before each selection of induced yeast, nanobody expression test were conducted with a
quick analytical flow cytometry assay achieved by Life Tech Attune NxT flow cytometer. First,
1x10
6
induced yeast and 500μL selection buffer (150mM NaCl, 25mM HEPES pH 7.5, 10%
glycerol, 1mM maltose) were added to two microcentrifuge tubes and both tubes were
centrifuged to obtain yeast pellets. Secondly, yeast pellets were resuspended in 100ul selection
buffer. Thirdly, one tube was added with ~0.5 μg of anti-HA rabbit antibody and ~0.5 μg of goat
anti-rabbit IgG labeled with APC and the other tube were left unstained as a control. Fourthly,
both tubes were rocked at 4 °C for 15 minutes followed by spinning down cells for 1 minute at
3500 x g, 4 °C. Fifthly, yeast pellets were resuspended in 100ul selection buffer and the yeast
with anti-HA antibody were added with ~0.5 μg of goat anti-rabbit IgG labeled with APC.
Sixthly, both tubes were rocked at 4 °C for 15 minutes again followed by spinning down cells for
1 minute at 3500 x g, 4 °C. Seventhly, cells were resuspended in 100 μl of selection buffer and
nanobody expression level were assessed on flow cytometer using unstained sample to set gates.
The maximum number of expressing cells from the naïve library is ~25%. The typical number of
expressing cells is ~15-20%. If expression is on the low end (~8-12%) consider increasing the
number of cells used for nanobody selection to compensate. For round 1 selection by MACS, a
suitable number of cells to have at least 10-fold over library diversity is needed, which is 5 × 10
9

cells for the naïve library (NbLib). In my round 1 selection by MACS, yeast expression level was
around 6% and 1x10
10
yeast cells were used for selection. Negative selection was performed
40
with yeast incubated in selection buffer containing 1uM Monoclonal ANTI-FLAG® M2
antibody produced in mouse (Millipore-Sigma, Burlington, MA) and 500ul Anti-Mouse IgG1
MicroBeads (Miltenyi Biotec, Gaithersburg, MD) with a total volume of 5mL. Positive selection
was performed with negative-selected yeast incubated in selection buffer containing 1.5uM
purified MCECTO, 1uM M2-Flag and 500ul Anti-Mouse IgG1 MicroBeads also with a total
volume of 1mL. After each round selection by MACS or FACS, yeast cells were recovered in -
Trp + glucose media followed by shaking for 24 hours, 30 °C, 220 rpm. In my round 2 selection
by MACS, 5x10
8
yeast cells were used for selection. Negative selection was performed with
yeast incubated in selection buffer containing 1uM Monoclonal ANTI-FLAG® M2 antibody
produced in mouse and 200ul Anti-Mouse IgG1 MicroBeads with a total volume of 1mL.
Positive selection was performed with negative-selected yeast incubated in selection buffer
containing 1.5uM purified MCECTO, 1uM M2-Flag and 200ul Anti-Mouse IgG1 MicroBeads
also with a total volume of 1mL. Both round 3 and round 4 selections by FACS used 1x10
8
cells.
Yeast cells were labeled by incubating with selection buffer containing 1uM purified MCECTO
and 660nM APC anti-DYKDDDDK(FLAG) Tag Antibody (Biolegend, San Diego, CA) with a
total volume of 348ul (cells for round 4 selection should be incubated in a smaller scale solution,
but I failed to follow this rule). Another 1x 10
7
cells were incubated with selection buffer only
containing 660nM APC anti-DYKDDDDK (FLAG) Tag Antibody with a total volume of 34.8ul.
Yeast cells expressing nanobodies against MCECTO were sorted at 0.06% (round 3) and at
0.74% (round 4) positive on a BD FACSAriaTM III flow cytometer using 10x smaller scale-
stained cells without purified MCECTO as a gate.

41
After round four selection, about 4x10
5
yeast cells were isolated and half of the cells were
plated on -Trp + glucose agar plates to isolate single clones after 3 days. Since pink-color
adenine-deficient yeast colonies were discovered on plates, normal white yeast colonies and pink
yeast colonies were randomly picked out with each group including 5 different colonies. To
check the difference of binding capability of two groups of yeast to MCECTO, 1x10
6
yeast cells
for each colony were labeled with 0.5ug purified FLAG-tagged MCECTO + 0.5ug PE anti-
DYKDDDDK (FLAG) Tag Antibody (Biolegend, San Diego, CA) in 100ul selection buffer on a
96-well round-bottom plate followed by measuring percentage of positive cells on Life Tech
Attune NxT flow cytometer using one well of cells without MCECTO as gate. It turned out that
only adenine-sufficient group showed relative binding capacity to MCECTO. Therefore, seventy
more adenine-sufficient single colonies were randomly picked out to check binding capability to
MCECTO. After obtaining all the results from the cell flow cytometer, 11 clones detected with
more than 9% fluorescent cells were characterized as having binding capability to MCECTO and
3 clones detected with more than 5% fluorescent cells were characterized as having moderate
binding capability to MCECTO. These 14 clones were chosen for sequencing and further
characterization.

Nanobody Sequencing
Yeast plasmids containing nanobody sequences were isolated by PCI (Phenol: Chloroform:
Isoamyl alcohol) method (Sigma-Aldrich, St. Louis, MO) and were transformed into TOP10
electrocompetent cells by electroporation and grown on LB-Ampicillin plates. Plasmids
containing nanobody genes were extracted from TOP10 cells and sequenced with Gal1 primer.
The 14 sequencing results of nanobodies from yeast plasmids were listed in the following table.
42
Sequence
number
Nanobody sequence
1 1
QVQLQESGGGLVQAGGSLRLSCAASGTISFGKYMGWYRQAPGKEREFVAAINGGTNTNYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAARKWRWLQHVYWGQGTQVTVSS 118
2 1
QVQLQESGGGLVQAGGSLRLSCAASGSIFDHVKMGWYRQAPGKEREFVAGIGWGATTNYADSVKG
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVNRDRWARHHYWGQGTQVTVSS 118
3 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFYATYMGWYRQAPGKEREFVAAIAIGSTTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKLYYGSRWTGNLPYWGQGTQVTVSS 122
4 1
QVQLQESGGGLVQAGGSLRLSCAASGSIFTWPRMGWYRQAPGKEREFVASIGRGSSTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYPYYVSSRSAKSSLSHYYWGQGTQVTVSS 126
5 1
QVQLQESGGGLVQAGGSLRLSCAASGTISPYYTMGWYRQAPGKEREFVASISSGGITYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAVVRQYKRDYDKVHDYWGQGTQVTVSS 122
6 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFHRWPMGWYRQAPGKEREFVAAITNGANTNYADSVKG
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARDYDQKTAWGDLYYWGQGTQVTVSS 122
7 1
QVQLQESGGGLVQAGGSLRLSCAASGYIFRQYPMGWYRQAPGKEREFVAAINLGGITNYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAVTRYRWRWLDAYIKRSFLYWGQGTQVTVSS 126
8 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFYATYMGWYRQAPGKEREFVAAIAIGSTTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKLYYGSRWTGNLPYWGQGTQVTVSS 122
9 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFLSGYMGWYRQAPGKEREFVAGINYGTITYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAARIWVGHRLYYWGQGTQVTVSS 118
10 1
QVQLQESGGGLVQAGGSLRLSCAASGSISRKDTMGWYRQAPGKEREFVASIAGGTTTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAVVDDHHYPYRYWGQGTQVTVSS 118
11 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFWGLSMGWYRQAPGKERELVAGISAGGITYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYKKyirRWFSDGIDDHYYWGQGTQVTVSS 126
12 1
QVQLQESGGGLVQAGGSLRLSCAASGNIFLSGYMGWYRQAPGKEREFVAGINYGTITYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAARIWVGHRLYYWGQGTQVTVSS 118
13 1
QVQLQESGGGLVQAGGSLRLSCAASGTISAYYGMGWYRQAPGKEREFVASIGRGSSTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAVGYRSTKYYWFLAYWGQGTQVTVSS 121
14 1
QVQLQESGGGLVQAGGSLRLSCAASGYISEPYRMGWYRQAPGKEREFVATIDFGGTTNYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAWTRLRTLAVIRLKYWGQGTQVTVSS 122

Cloning and Primers
Cloning was performed using the restriction enzyme ligation method and the Gibson
assembly method. For cloning mouse MCECTO into pFUSEN-mG2AFc (InvivoGen, San Diego,
CA), the restriction enzyme ligation method was used. All inserts were designed to contain
restriction enzyme ligation sites at both ends. Restriction endonucleases were chosen based on
whether Fc structure on the vector was removed or reserved. When Fc was intended to be
43
removed, the vector was linearized by EcoRI and BamHI digestion. Two inserts were designed
including a FLAG tag either at front or at rear of MCECTO. The inserts were amplified by PCR
and were also digested by the same restriction endonucleases used for the vector. Then either
recombinant was created by DNA ligase covalently linking the vector and the insert. When Fc
was intended to be reserved, an insert was designed for mouse MCECTO with a PreScission
cleavage site at its front and a FLAG tag at its rear. The vector and the insert were digested by
BamHI and NheI. For cloning human MCECTO into pFUSEN-hG1Fc plasmid (InvivoGen, San
Diego, CA), an insert was designed the same as mouse MCECTO cloning when Fc was intended
to be reserved. The same restriction enzymes were also used. For cloning nanobodies into
periplasm expression vector pET-26b with a C terminal 6 histidine tag, the Gibson assembly
method was used. The vector was linearized by NcoI and XhoI digestion. Nanobody sequences
were PCR amplified from yeast and the Gibson method was used to assemble the fragments.
Primers used are listed in the following table.
EcoRI-Flag-mMCECTO-F GATCGAATTCGGATTACAAGGATGACGACGATAAGAAAAA
TTCTGAGATGTCCAAG
mMCECTO-STOP-BamHI-R CTAGGGATCCTTATGTACTGGGCTGAGGCTGTGC
EcoRI-mMCECTO-F GATCGAATTCGAAAAATTCTGAGATGTCCAAG

mMCECTO-Flag-STOP-BamHI-R CTAGGGATCCTTACTTATCGTCGTCATCCTTGTAATCTGTAC
TGGGCTGAGGCTGTGC
BamHI-PreS-mMCECTO-F GATCGGATCCCTGGAAGTTCTGTTCCAGGGGCCCCTGAAAA
ATTCTGAGATGTCCAAG
mMCECTO-Flag-STOP-NheI-R CTAGGCTAGCTTACTTATCGTCGTCATCCTTGTAATCTGTAC
TGGGCTGAGGCTGTGC
BamHI-PreS-hMCECTO-F GATCGGATCCCTGGAAGTTCTGTTCCAGGGGCCCCTGAAGA
ATGCTGAGATGTCCAAG
hMCECTO-Flag-STOP-NheI-R CTAGGCTAGCTTACTTATCGTCGTCATCCTTGTAATCTTGAG
GTGAGGACTGTGGCAT
NcoI-Nanobody-F GCTGCCCAGCCGGCGATGGCCCAGGTGCAGCTGCAGGAAAG
Nanobody-his-XhoI-R AGTGGTGGTGGTGGTGGTGCTCAGTGGTGGTGGTGGTGGC
TGCTCACGGTCACCTG
44
References
1. Aldenborg, F., & Enerbäck, L. (1988). Histochemical heterogeneity of dermal mast cells in
athymic and normal rats. The Histochemical Journal, 20(1), 19–28.
https://doi.org/10.1007/BF01745965

2. Alfaleh, M. A., Alsaab, H. O., Mahmoud, A. B., Alkayyal, A. A., Jones, M. L., Mahler, S. M.,
& Hashem, A. M. (2020). Phage Display Derived Monoclonal Antibodies: From Bench
to Bedside. Frontiers in Immunology, 11, 1986.
https://doi.org/10.3389/fimmu.2020.01986

3. Almagro, J. C., Pedraza-Escalona, M., Arrieta, H. I., & Pérez-Tapia, S. M. (2019). Phage
Display Libraries for Antibody Therapeutic Discovery and Development. Antibodies,
8(3), 44. https://doi.org/10.3390/antib8030044

4. Aricescu, A. R., & Owens, R. J. (2013). Expression of recombinant glycoproteins in
mammalian cells: Towards an integrative approach to structural biology. Current
Opinion in Structural Biology, 23(3), 345–356. https://doi.org/10.1016/j.sbi.2013.04.003

5. Chipps, B. E., Figliomeni, M., & Spector, S. (2012). Omalizumab: An update on efficacy and
safety in moderate-to-severe allergic asthma. Allergy and Asthma Proceedings, 33(5),
377–385. https://doi.org/10.2500/aap.2012.33.3599

6. Correa, A., & Oppezzo, P. (2015). Overcoming the Solubility Problem in E. coli: Available
Approaches for Recombinant Protein Production. In E. García-Fruitós (Ed.), Insoluble
Proteins: Methods and Protocols (pp. 27–44). Springer. https://doi.org/10.1007/978-1-
4939-2205-5_2

7. Crivellato, E., Beltrami, C. A., Mallardi, F., & Ribatti, D. (2003). Paul Ehrlich’s doctoral
thesis: A milestone in the study of mast cells. British Journal of Haematology, 123(1),
19–21. https://doi.org/10.1046/j.1365-2141.2003.04573.x

8. Dahlin, J. S., & Hallgren, J. (2015). Mast cell progenitors: Origin, development and migration
to tissues. Molecular Immunology, 63(1), 9–17.
https://doi.org/10.1016/j.molimm.2014.01.018

9. Elieh Ali Komi, D., Wöhrl, S., & Bielory, L. (2020). Mast Cell Biology at Molecular Level: A
Comprehensive Review. Clinical Reviews in Allergy & Immunology, 58(3), 342–365.
https://doi.org/10.1007/s12016-019-08769-2

10. Ferrer-Miralles, N., Saccardo, P., Corchero, J. L., Xu, Z., & García-Fruitós, E. (2015).
General Introduction: Recombinant Protein Production and Purification of Insoluble
Proteins. In E. García-Fruitós (Ed.), Insoluble Proteins: Methods and Protocols (pp. 1–
24). Springer. https://doi.org/10.1007/978-1-4939-2205-5_1

45
11. Galli, S. J., Gaudenzio, N., & Tsai, M. (2020). Mast Cells in Inflammation and Disease:
Recent Progress and Ongoing Concerns. Annual Review of Immunology, 38(1), 49–77.
https://doi.org/10.1146/annurev-immunol-071719-094903

12. Galli, S. J., & Tsai, M. (2012). IgE and mast cells in allergic disease. Nature Medicine, 18(5),
693–704. https://doi.org/10.1038/nm.2755

13. Galli, S. J., Tsai, M., & Wershil, B. K. (1993). The c-kit receptor, stem cell factor, and mast
cells. What each is teaching us about the others. The American Journal of Pathology,
142(4), 965–974.

14. Ghably, J., Saleh, H., Vyas, H., Peiris, E., Misra, N., & Krishnaswamy, G. (2015). Paul
Ehrlich’s Mastzellen: A Historical Perspective of Relevant Developments in Mast Cell
Biology. In M. R. Hughes & K. M. McNagny (Eds.), Mast Cells: Methods and Protocols
(pp. 3–10). Springer. https://doi.org/10.1007/978-1-4939-1568-2_1

15. Gilfillan, A. M., Austin, S. J., & Metcalfe, D. D. (2011). Mast Cell Biology: Introduction and
Overview. In A. M. Gilfillan & D. D. Metcalfe (Eds.), Mast Cell Biology: Contemporary
and Emerging Topics (pp. 2–12). Springer US. https://doi.org/10.1007/978-1-4419-9533-
9_1

16. Gilfillan, A. M., & Tkaczyk, C. (2006). Integrated signalling pathways for mast-cell
activation. Nature Reviews Immunology, 6(3), 218–230. https://doi.org/10.1038/nri1782

17. Harmsen, M. M., & De Haard, H. J. (2007). Properties, production, and applications of
camelid single-domain antibody fragments. Applied Microbiology and Biotechnology,
77(1), 13–22. https://doi.org/10.1007/s00253-007-1142-2

18. Kalesnikoff, J., & Galli, S. J. (2008). New developments in mast cell biology. Nature
Immunology, 9(11), Article 11. https://doi.org/10.1038/ni.f.216

19. Kitamura, Y. (n.d.). Heterogeneity of Mast Cells and Phenotypic Change Between
Subpopulations. 18.

20. Köhler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature, 256(5517), 495–497. https://doi.org/10.1038/256495a0

21. Kolkhir, P., Elieh-Ali-Komi, D., Metz, M., Siebenhaar, F., & Maurer, M. (2021).
Understanding human mast cells: Lesson from therapies for allergic and non-allergic
diseases. Nature Reviews. Immunology. https://doi.org/10.1038/s41577-021-00622-y

22. Leurs, R. (2011). En route to new blockbuster anti-histamines: Surveying the offspring of the
expanding histamine receptor family. 32(4), 8.

23. Li, K., Wang, S.-W., Li, Y., Martin, R. E., Li, L., Lu, M., Lamhamedi-Cherradi, S.-E., Hu,
G., Demissie-Sanders, S., Zheng, J., Chung, F., Oates, T., & Yao, Z. (2005).
46
Identification and expression of a new type II transmembrane protein in human mast
cells. Genomics, 86(1), 68–75. https://doi.org/10.1016/j.ygeno.2005.03.006

24. Maurer, M., Rosén, K., Hsieh, H.-J., Saini, S., Grattan, C., Gimenéz-Arnau, A., Agarwal, S.,
Doyle, R., Canvin, J., Kaplan, A., & Casale, T. (2013). Omalizumab for the Treatment of
Chronic Idiopathic or Spontaneous Urticaria. New England Journal of Medicine, 368(10),
924–935. https://doi.org/10.1056/NEJMoa1215372

25. McMahon, C., Baier, A. S., Pascolutti, R., Wegrecki, M., Zheng, S., Ong, J. X., Erlandson, S.
C., Hilger, D., Rasmussen, S. G. F., Ring, A. M., Manglik, A., & Kruse, A. C. (2018).
Yeast surface display platform for rapid discovery of conformationally selective
nanobodies. Nature Structural & Molecular Biology, 25(3), 289–296.
https://doi.org/10.1038/s41594-018-0028-6

26. Mullard, A. (2021). FDA approves 100th monoclonal antibody product. Nature Reviews
Drug Discovery, 20(7), 491–495. https://doi.org/10.1038/d41573-021-00079-7

27. Muyldermans, S. (2013). Nanobodies: Natural Single-Domain Antibodies. Annual Review of
Biochemistry, 82(1), 775–797. https://doi.org/10.1146/annurev-biochem-063011-092449

28. Nelson, A. L. (2010). Antibody fragments: Hope and hype. MAbs, 2(1), 77–83.
https://doi.org/10.4161/mabs.2.1.10786

29. Nettleship, J. E., Watson, P. J., Rahman-Huq, N., Fairall, L., Posner, M. G., Upadhyay, A.,
Reddivari, Y., Chamberlain, J. M. G., Kolstoe, S. E., Bagby, S., Schwabe, J. W. R., &
Owens, R. J. (2015). Transient Expression in HEK 293 Cells: An Alternative to E. coli
for the Production of Secreted and Intracellular Mammalian Proteins. In E. García-
Fruitós (Ed.), Insoluble Proteins: Methods and Protocols (pp. 209–222). Springer.
https://doi.org/10.1007/978-1-4939-2205-5_11

30. Raman, K., O’Donnell, M. J., Czlonkowska, A., Duarte, Y. C., Lopez-Jaramillo, P.,
Peñaherrera, E., Sharma, M., Shoamanesh, A., Skowronska, M., Yusuf, S., & Paré, G.
(2016). Peripheral Blood MCEMP1 Gene Expression as a Biomarker for Stroke
Prognosis. Stroke, 47(3), 652–658. https://doi.org/10.1161/STROKEAHA.115.011854

31. Rastogi, D., Nico, J., Johnston, A. D., Tobias, T. A. M., Jorge, Y., Macian, F., & Greally, J.
M. (2018). CDC42-related genes are upregulated in helper T cells from obese asthmatic
children. Journal of Allergy and Clinical Immunology, 141(2), 539-548.e7.
https://doi.org/10.1016/j.jaci.2017.04.016

32. Ryu, H., Fuwad, A., Yoon, S., Jang, H., Lee, J. C., Kim, S. M., & Jeon, T.-J. (2019).
Biomimetic Membranes with Transmembrane Proteins: State-of-the-Art in
Transmembrane Protein Applications. International Journal of Molecular Sciences,
20(6), E1437. https://doi.org/10.3390/ijms20061437

47
33. Su, M.-W., Lin, W.-C., Tsai, C.-H., Chiang, B.-L., Yang, Y.-H., Lin, Y.-T., Wang, L.-C.,
Lee, J.-H., Chou, C.-C., Wu, Y.-F., Yeh, Y.-L., & Lee, Y. L. (2018). Childhood asthma
clusters reveal neutrophil-predominant phenotype with distinct gene expression. Allergy,
73(10), 2024–2032. https://doi.org/10.1111/all.13439

34. Subrata, L. S., Bizzintino, J., Mamessier, E., Bosco, A., McKenna, K. L., Wikström, M. E.,
Goldblatt, J., Sly, P. D., Hales, B. J., Thomas, W. R., Laing, I. A., LeSouëf, P. N., &
Holt, P. G. (2009). Interactions between Innate Antiviral and Atopic
Immunoinflammatory Pathways Precipitate and Sustain Asthma Exacerbations in
Children. The Journal of Immunology, 183(4), 2793–2800.
https://doi.org/10.4049/jimmunol.0900695

35. Thomas, P., & Smart, T. G. (2005). HEK293 cell line: A vehicle for the expression of
recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51(3),
187–200. https://doi.org/10.1016/j.vascn.2004.08.014

36. Virk, H., Arthur, G., & Bradding, P. (2016). Mast cells and their activation in lung disease.
Translational Research, 174, 60–76. https://doi.org/10.1016/j.trsl.2016.01.005

Abstract (if available)

Abstract Mast cells and their activation can be detrimental to human health. KIT is one of the most important receptors for the proliferation and activation of mast cells. Reducing mast cell numbers and preventing mast cell differentiation by inhibiting KIT receptor has been proved to be a good approach to treat mast cell-driven diseases as evinced by currently approved drugs. We hypothesize that MCEMP1 positively regulates KIT receptor activation and contributes to the development of pulmonary diseases like asthma. Herein, I aim to identify a MCEMP1-specific nanobody that can be used not only as a research tool to study a biological function of MCEMP1, but also as a potential therapeutic agent. In this study, HEK 293T cells were utilized to produce the ectodomain of MCEMP1 (MCECTO) of mouse and the antigen-specific nanobodies were screened with a synthetic yeast display nanobody library by magnetic cell sorting (MACS) and fluorescence activated cell sorting (FACS) . I obtained 2.756mg of the protein MCECTO in total and sorted out 0.74% of positive yeast cells targeting antigen MCECTO. I identified 11 yeast colonies with >9% positive rates and 3 colonies with >5% positive rates of MCECTO-binding affinity out of 75 different adenine-sufficient yeast colonies with a quick analytical flow cytometry assay. 3 nanobody candidates were selected to further characterize for mouse MCEMP1-specific inhibitory nanobodies after sequencing analysis.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer immunotherapy

PDF

Self-secretion of checkpoint blockade enhances antitumor immunity by murine chimeric antigen receptor-engineered T cells

PDF

SARS-CoV-2 interaction with the CD1d/NKT cell antigen presentation system

PDF

SARS-CoV-2 suppression of CD1d expression and NKT cell function

PDF

Cytotoxicity and total ROS generation of insoluble nickel (II) compounds of beas-2B cells: role of reactive oxygen species (ROS)

PDF

Regulation of T cell HLA-DR by CD3 ζ signaling

PDF

The essential role of histone H2A deubiquitinase MYSM1 in natural killer cell maturation and HSC homeostasis

PDF

Molecular mechanism for the immune evasion of CD1d antigen presentation by herpes simplex virus-1 UL56 protein

PDF

Tri-specific T cell engager immunotherapy targeting tumor initiating cells

PDF

Modeling SynGAP1 truncating mutations in neurodevelopmental disease using iPSC-derived neurons

PDF

CAD-mediated metabolic reprogramming is regulated by innate immune kinases TBK1 and IKKβ

PDF

The role of envelope protein in SARS-CoV-2 evasion of CD1d antigen presentation pathway

PDF

Virus customization of host protein machinery for efficient propagation

PDF

Beclin 2-mediated autophagic degradation of the pathogenic proteins in neurodegenerative diseases

PDF

PD-L1-GM-CSF fusion protein-loaded DC vaccination activates PDL1-specific humoral and cellular immune responses

PDF

Mechanistic insights into HSV-1 UL56-mediated immune evasion through CD1d downregulation and NKT cell suppression

PDF

Development of an NFAT-GFP Jurkat T cell reporter system for acceleration of T Cell receptor affinity screening

PDF

Tri-specific T cell engager immunotherapy targeting tumor initiating cells

PDF

Novel approaches of mobilizing human iNKT cells for cancer immunotherapies

PDF

Comparative analysis of scFv and non-scFv based chimeric antigen receptors (CARs) against B cell maturation antigen (BCMA)

Asset Metadata

Creator Xiong, Yu (author)

Core Title Yeast-based nanobody identification for MCEMP1: mast-cell expressed membrane protein 1

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2022-12

Publication Date 12/06/2024

Defense Date 10/06/2022

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

Tag MCEMP1,nanobody,OAI-PMH Harvest,pulmonary diseases

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Akbari, Omid (committee chair), Chen, Siyi (committee member), Landolph, Joseph (committee member), Yuan, Weiming (committee member)

Creator Email xiongyu@usc.edu,xiongyu735@gmail.com

Unique identifier UC112617592

Identifier etd-XiongYu-11343.pdf (filename)

Legacy Identifier etd-XiongYu-11343

Document Type Thesis

Format theses (aat)

Rights Xiong, Yu

Internet Media Type application/pdf

Type texts

Source 20221207-usctheses-batch-994 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu