Thesis Project Luke Morton 2016

Serine Proteases, A Basis of Immunity
Through Evolution
Luke Morton
Degree project in biology, Master of science (2 years), 2016
Examensarbete i biologi 45 hp till masterexamen, 2016
Biology Education Centre
Supervisor: Lars Hellman
External opponent: Srinivas Akula

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Table Of Contents
• Abstract…………………………………………………………..3
• Introduction……………………………………………………...4
• Materials & Methods……………………………………………9
o Bacterial Transformation & multiplication
o HEK 293 Growth, Transfection & Expression
o Quantification of Protein
o Chromogenic Substrate Analysis
o Recombinant Substrate Analysis
o Phage Display Substrate Analysis
• Results……………………………………………………………18
o Expression of Proteases
o Bradford Assay & Quantification with BSA Standard
o Activation with Enterokinase
o Recombinant Substrate Analysis
o Chromogenic Substrate Analysis
o Phage Display
• Discussion………………………………………………………..29
• Acknowledgements
• References

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Abstract
Serine proteases are one of the most well studied enzyme families in
mammals. However, this does not, translate directly to a deeper understanding of the
development and appearance of these essential components of life during vertebrate
evolution. Serine proteases are found throughout the entire animal kingdom because of
their importance in immunity, food digestion, blood coagulation and mechanisms of
transport and invasion in bacteria and viruses. One subfamily of these serine proteases
are stored in granules in cells of our immune system and they’re responsible for immune
defense. Cells of our immune system that store such proteases are neutrophils, mast cells,
cytotoxic T cells and NK cells. These proteases have quite diverse primary specificities
including tryptase, chymase, elastase, asp-ase and met-ase specificities. One central
question is here, how did they appear as such a specialized force against the infectious
organisms of our environment? Using bioinformatic databases to establish structural
relationships, a map of genetic homology can be constructed, illuminating the origin and
diversification of some of the most vital components of biological systems. Many of
these proteases can provide pivotal information of the evolutionary puzzle, but their
function must first be elucidated. To look deeper into the evolution of these proteases we
decided to study the specificity of two non-mammalian hematopoietic serine proteases
the chicken CTSG and Chinese alligator MCP-1. Their coding regions were first
synthesized as designer genes and then transfected into mammalian cells for expression
and analysis of their extended cleavage specificity. This was the first step in an attempt
to provide important information of the transition of immune defense from birds and
reptiles to mammals. This report is an attempt to connect bioinformatic data with
experimental data of these two proteases; a step-by-step approach using molecular
methods to bridge the gap between genetic similarity and functional reality.

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Introduction
As the primordial pool began to reveal single celled organisms, the war for
survival began. The persistent arms race between organism and its environment lends the
evolutionary journey through immune system development a chaotic, but impressive
history. As these organisms have absorbed or formed symbiotic relationships,
compounding complexities arose. Convergent and divergent evolutionary pathways,
duplication of genes and random mutations created and separated species over millions of
years. The mechanisms of specific processes are conserved or abandoned based on
environmental pressure. Some of these highly conserved mechanisms have led to the
successful generation of functional serine proteases, enzymes that are very important in
many biological processes and also essential for a functioning immune system. Keeping
track of enzymatic specificity and preferred targets not only allows us to trace lineages
back to significant historical separations, but learn what shaped our immune system and
help explain our own mechanisms of defense (1).
Serine proteases are abundant in all vertebrates; members of the family are found
in cartilaginous fish and can be traced back into bacterial models (2,3). The evolution of
the serine protease family from these bacterial strains to modern day primates is still
vastly unexplored. Evolutionary studies dealing with relatively small biologically active
peptides can be extremely difficult because of high conservation (4), however; with the
development of sequencing techniques and exponentially growing databases of
sequenced organisms, a comparative analysis can begin to explain synteny (5). To discern
evolution in an immunological perspective, focusing on proteases can give important new
insights.
Within the 560-protease genes present in primates 150 are serine proteases, of
which 50% are chymotrypsin related (6). A subfamily of these chymotrypsin related
serine proteases are expressed by hematopoietic cells and these are in mammals in four
different chromosomal loci: two tryptase loci, one for mast cells and the other for T-cells,
a met-ase locus and the chymase loci (7). Within the frame of this report, focus will be
on the chymase locus homologs of chicken and Chinese alligator, more specifically the
chicken cathepsin G (CTSG) and the alligator mast cell protease 1 (MCP-1) serine
proteases. These two proteases, once the cleavage specificity is better understood will

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pave the way for illuminating the chymase or tryptase locus transition into mammals
from reptiles and birds.
With a growing array of online resources, we have used MrBayes algorithms to
determine homology and potential evolutionary descendants of members of the large
family of hematopoietic serine proteases (8). The evolutionary map generated from this
type of studies can aid us in deciding the next steps in our analysis of their evolution.

Figure 1. Chicken CTSG & Chinese Alligator MCP-1 Loci
Chicken CTSG and Alligator MCP-1 locations compared to Human Chymase.
Through genetic sequence similarity, these chromosomal regions provide genes for the two proteases of
study; similar surrounding genes that are conserved through many other species, lends weight to serine
protease identification and activity, establishing these locations.
(Adopted from Akula & Hellman 2015)
The oldest granule associated serine proteases involved in immune defense are the
granzymes A and K, which are found as far back as the cartilaginous fish. The met-ase
locus seems to materialize in the bony fish, with similar bordering genes as in mammals
(8). A clearly defined chymase locus has only been found in placental and marsupial
mammals. However, although no similar bordering genes were found in Chinese
alligator and chicken loci, homologous contigs were present (8). These two enzymes have
here been studied in order to understand the evolution of the chymase locus and the genes
found within this locus. The aim was to determine extended cleavage specificities of
these two enzymes. Chicken CTSG, which has a triplet reminiscent of a tryptase is found
on chromosome 28 and exists in a locus that only shows similar synteny with other
reptiles and birds. Chinese alligator MCP-1 was located, based on gene homology, on an
unknown chromosome but is found on a locus that most closely resembles the chymase
locus in mammals (Fig. 1) (8). These two enzymes could, if cleavage specificity is as
predicted, illuminate chymase locus development back to early reptiles and birds.

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Figure 2. Locus Relationships in Phylogenetic Trees of Chicken CTSG & Alligator MCP-1
These trees are made based on an algorithm that compares sequence homology and produces evolutionary
maps. (Adopted from Akula & Hellman 2015)
Proteases of this class are important granule constituents of hematopoietic cells
namely neutrophils, cytotoxic T-cells, mast cells and NK cells. They have a number of
different functions of which some are known and others that are still being elucidated.
The proteases found in these granules have mostly immunological and migratory
functions (degradation of pathogen/host membranes or antimicrobial activity) but other
members of this large family have also functions in blood coagulation, food digestion,
homeostasis and fertilization (7).
Within the granulocytes, the granules containing these proteases can fuse with
lysozomes where both reactive oxygen species and the enzymes work in concert to
degrade and kill pathogens, or can be released into the surrounding tissue to act as
inflammatory mediators (9). Many serine proteases have also been linked to connective
tissue remodeling in cases of inflammation (10). The chicken CTSG and alligator MCP-1
have got names based on their homology to various mammalian enzymes and have most
likely roles in immune defense and granulocyte migration.
The mode of action is dependent on the catalytic triad. This is the active site,
made up of a histadine at position 57, an aspartic acid at position 102 and the highly
reactive serine at position 195, lending its name to the class of protease (Fig 6.) (11). This
catalytic triad is responsible for the hydrolysis of the substrate through proton transfer
between these three amino acids.

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Figure 3. Detailed Phylogenetic Tree of Serine Protease Loci
The aligment was based on the MrBayes algorithm
(Taken from Akula & Hellman 2015)

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The selection of which amino acid the enzyme prefers in the cleavage pocket is also
determined by three amino acids. These are found in positions 189, 216 and 226 (S1
pocket) according to the chymotrypsin numbering, with the 189th
position being the most
critical (Fig. 4) (12). Chymases have a triplet of small aliphatic amino acids, which allow
for larger hydrophobic aromatic amino acids like phenylalanine, tyrosine and tryptophan
to enter that pocket. Tryptases generally have negatively charged triplets that will favor
basic amino acids likes arginine and lysine in the S1 pocket (Fig. 4).
Serine proteases are one of the most actively studied enzymes throughout the
human genome because of their diverse array of functions (2, 12). As their cleavage
specificities and evolutionary relationships become better known, conclusions can be
drawn, and therapeutic avenues can be explored with greater confidence. The seemingly
backward trajectory we take in this study may not immediately grant applicable medical
results but is essential for our understanding of their appearance and diversification
during vertebrate evolution, which in turn could give important insight into the evolution
of our own immune mechanisms. The aim of this study is to elucidate the extended
cleavage specificity of chicken cathepsin G (CTSG) and Chinese alligator mast cell
protease-1 (MCP-1) with various laboratory techniques to obtain a more detailed picture
of the early events in the formation of the mammalian chymase locus and their genes and
enzyme specificities.

Figure 4. S1 Pockets of The Different Proteases
First, the chymotrypsin referred to as the chymase has small aliphatic amino acids in the S1 pocket
allowing for bulky aromatic amino acids to fit and subsequently be cleaved. The trypsin S1 pocket has an
aspartic acid in the 189 position favoring basic amino acid attraction and cleavage. Third, the elastase, not
wholly mentioned in this correspondence, favors smaller and a broader range of amino acids because of its
threonine in position 226. (Adopted from Hellman & Thorpe 2014)

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Figure 5. Cleavage Nomenclature and Position
A serine protease in the chymotrypsin-numbering scheme will have a triplet that dictates cleavage
specificity; this is commonly referred to as the S1 pocket, the corresponding location on a protein
(substrate) in which it cleaves is labeled in front and behind the site of cleavage by P or P (prime),
allowing for quick reference to modes of action and classification.
(Adopted from Hellman & Thorpe 2014)

Figure 6. The Catalytic Triad Proton Transfer
The Serine in position 195 gives the proteases their name and is responsible for the attack on the
substrate’s carbonyl carbon that facilitates proton transfer to the histadine 57 then to the Aspartic acid 102.
The creation of the oxyanion pocket via surrounding hydrogen bonding allows for stabilization of the
tetrahedral intermediate. This allows the proton movement back from the Asp 102 to the Ser 159 in the
second half of the reaction with the addition of water from the surrounding aqueous environment to cleave
the substrate from the 195 serine, completing the process. (Adopted from Hedstrom et al. 2003)

Materials & Methods
Transformation, multiplication and quantification of Pcep-Pu2 Vector within
Escherichia coli strain DK1.
Once the sequences of the chicken CTSG and alligator MCP-1 proteases had been
defined through bio-informatics (8) they were ordered from Genescript. Upon arrival the
inserts containing the coding regions for these two proteases were cloned into the pCEP-
Pu2 mammalian expression vector. We received a small amount of the plasmid that we
needed to amplify by transformation into the DK1 strain of E.coli. The DK1 strain is well

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known for its reliable replication machinery due to the lack of several recombinases. An
inoculum of an empty (vector-less) DK1 E.coli was taken from a freeze culture, which
contains 50% bacteria in LB and glycerol, stored at -80° Celsius, to an overnight culture
of 10 ml LB.

Adopted from Extended Substrate Specificity of Mast Cell α-chymases From Human and Dog by Maria
Fors 2013. Information on Pcep-Pu2 vector from Kohfeldt et al. (13)

The next day the 10ml overnight culture was added into 90ml LB and put into a
37°C room and shaken for about one and a half hours. Once the optical density (OD)
reached 0.5 the bacteria were put on ice then centrifuged at 4°C at 1.5 G into a pellet
while the supernatant is discarded. This pellet is resuspended in 10ml MgCl2 and again,
centrifuged to pellet. The pellet was then resuspended in 6ml of CaCl2, and then left on
ice for 30 minutes. The bacteria are now competent. Transformation is achieved with the
addition of 20ul of DNA pCEP-Pu2 vector with CTSG or MCP-1 insert to 200ul of
competent DK1 bacteria, incubation on ice for 30 minutes, add 100ul of LB then plate on
100uM ampicillin (AMP) plates after 1hr incubation. Bacteria cultured on these
ampicillin plates should have our construct that conveys resistance against the antibiotic.
DK1 cultures are taken and expanded and DNA is removed and purified using the Spin
Miniprep Kit.
Figure 7. Pcep-Pu2 Vector for
Mammalian Expression
Vector used in HEK 293 cell
line episomal expression.
Vector contains ampicillin
resistance as well as puromycin
resistance genes for selection.
Epstein-Barr nuclear antigen
(EBNA-1) provides stabilization
of the cell in the altered state.
Col E1 is a copy number
regulation system; preventing
plasmid collapse Also contains
various promoter regions (Ori
P, P CMV & SV40 pA). SV40 is
viral protein important for
consistent expression.

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To make sure our bacteria had the vector and required insert; a double restriction
enzyme cleavage reaction was run and shown on agarose gel. The vector itself has 2
restriction enzyme sites on either side of the sequence of interest: EcoRI and XhoI. In a
10ul cleavage reaction 3ul of deionized water, 1ul of 10x REB buffer, 5ul of our mini-
prep DNA, and 0.5ul of both restriction enzymes: EcoRI & XhoI, which are then
incubated for 2 and a half hours at 37°C. 10ul of DNA solution from cleavage is then
added to 2ul of 5x Ficoll Buffer (12.5g of Ficoll 400, 0.125g of Bromophenol blue,
0.125g of Xylencyanol & 400ul of 0.5M EDTA in 50ml dH2O). These samples are then
run on 1% agarose gel electrophoresis. The gel is made based on samples to be run and
the general size of those samples, 1% being appropriate for 500bp to 10kb.
Growth/Transfection & Expression in the HEK 293 cell line
(Information on HEK 293 cell lines from 14,15)
The DNA from the mini-preps needs to undergo ethanol precipitation before
transfection into HEK 293 cells. To the DNA solution add 1/10th
total volume 3M sodium
acetate (NaAc), and 2 volumes of 99% ethanol (EtOH), rotate tube to mix then freeze for
30 minutes. Centrifuge at max speed 14000 rpm for 10 minutes and remove the
supernatant then air-dry pellet and resuspend in 1/3rd
original volume in sterile TE
(10mM Tris pH7.5, 1mM EDTA in dH2O).
The HEK 293 stocks are kept at -80°C in a specific freeze-medium (DMEM
glutaMAX, 5% DMSO, 10% FBS and 50ug/ml gentamicin) and cultured in Dulbecco’s
modified Eagle’s medium (DMEM, GIBCO, Paisley, UK) with 10% fetal bovine serum
(FBS) and 50ug/ml gentamicin. First, cells were grown in a 25cm2
flask to about 70%
confluency. One 1.5ml Eppendorf tube is then filled with 10ul of precipitated mini-prep
DNA with 220ul of DMEM with gentamicin and 20ul of P3000 reagent. The second tube
is filled with 230ul DMEM with gentamicin plus 20ul of lipofectamine 3000 (a
transfection enhancer). Mix both tubes together and then add another 500ul of DMEM
w/gentamicin and vortex for 2 minutes. Leave at room temperature (RT) for 5 minutes
then add to a 14ml falcon tube containing 6ml of FBS free DMEM w/gentamicin. It is
important to wash the HEK 293 cells with DMEM w/gentamicin twice then add the
transfection mixture (total 7ml) and incubate at 37°C overnight. The next day add the

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10% FBS and leave overnight. There will be massive cell death (selection) but continue
to replace selection media with FBS until cells are again 70% confluent. With chicken
CTSG and alligator MCP-1 once confluence was reached it was time to expand them into
bigger flasks to increase the protein production. This just consists of shaking cell layer in
media dislodging cells and adding cell media mix to a 75cm2
then 175cm2
flask and
supplementing more DMEM to keep cells submerged. As the cells use the media and
produce waste and synthesized proteins, the media itself will turn yellow. Depending on
the population of cells the media will be collected as it begins to turn yellow and purified
for our protein of interest.
Filtrate the collected conditioned media through Munktell filter paper and then
into centrifuge bottles. Add 140ul of Ni-NTA slurry per 100ml of filtered media and
rotate in the cold room (4°) for 45 minutes. Prepare a 2-3ml syringe (w/o needle) with
glass fiber filter. Centrifuge at 1.5G and transfer beads to syringe after removing
supernatant. Wash beads with 1ml PBS tween 0.05% and 10mM imidazole three times.
Next is the elution step, which uses PBS tween 0.05%, 100mM imidazole. Six fractions
are collected; 1st
150ul and 2nd
-6th
at 300ul. These fractions are then run on Sodium
Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) to test concentration.
Quantification of Protein Concentration
The purity and concentration was determined based on the SDS-PAGE and
Bradford assays. Three separate dilutions of the protein are made and, depending on
perceived concentration, in this case, 1.1, 1:2, 1:5 was used. The Bradford reagents come
prepackaged from Bio-Rad, United States; reagent A, S and B. Combine reagent A and S
in a 50:1 ratio respectively with enough combined volume to cover 125ul per diluted
sample and a blank to calibrate the spectrophotometer. Add 125ul of A’ (A+S) to 25ul of
protein sample and vortex. Add 1mL of reagent B to each sample and incubate at room
temperate (RT) for 15 minutes and measure OD values at 405nm. These optical values
are then used in a predetermined equation (y=50.724x3
-1.8662x2
+2.6431x+0.0035) as x-
values to get y-values. Y-values are then multiplied with the dilution factors (1:2,1:5 and
no dilution) and averaged to get protein concentration.

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Activation and Testing Enzyme Specificity
Once the enzyme has been isolated it is activated with 1-2ul of enterokinase,
which cleaves off the histadine tag and the five amino acid enterokinase cleavage site.
This cleavage reaction is performed during 2 hours at 37°C with 1.5ul of enterokinase per
100ul of enzyme ranging from 0.3ug/ul to 1ug/ul concentrations.
Initial Recombinant Substrate Analysis
Since the triplet of chicken CTSG was known and indicated a tryptase activity, an
initial set of recombinant substrates from previous catfish studies were tried to see if it
had tryptase activity. The alligator triplet gave little guidance but was tested with the
same substrates. These substrates are eight amino acid long residues that are ordered
oligos (short peptide sequences), which are then ligated into an expression pET21a vector
and expressed in the bacterial Rosetta Gami E.coli T7 expression system. The oligo was
ligated between two copied of the bacterial redox protein thioredoxin in a recombinant
enzyme system named the 2xTRX gene ssyetm that has been used to study the extended
specificity of a number of serine proteases. Three previously produced varaints for a
Catfish enzyme, variants 1, 2 and 3 were used first because of that they encode multiple
arginines and a methionine in the case of V1, around the P1 position. Substrates ordered
in oligopeptide form from Sigma Aldrich.
• Catfish V1: RVTGMSLV 6ul substrate in 34ul PBS with 10ul enzyme
• Catfish V2: VVRRAAAG 7ul substrate in 38ul PBS with 5ul enzyme
• Catfish V3: VVRRRAAG 4.5ul substrate in 40.5 PBS with 5ul enzyme
For a recombinant substrate assay a master mix is made containing 5ug/10ul of
protein with a final volume of 50ul for each time point: 0min, 15min, 45min and 150min.
Four tubes are then labeled with these time points and filled with 2.5ul of 4x LDS buffer
for SDS-PAGE that chelates the enzymatic action and stops interaction with substrate.
Three different chymase substrate variants were later used in a recombinant substrate
assay:

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• Human Chymase Consensus: VVLFSEVL 13.5ul substrate in 26.5ul PBS with
5ul MCP-1.
• Opossum Chymase Consensus: VGLWLDRV 13.5ul substrate in 26.5ul PBS
with 5ul MCP-1.
• Human Chymase Variant Six: VVLLSEVL 6.5ul substrate in 38.5ul PBS with
5ul MCP-1.
Three Granzyme B substrates were tried with alligator MCP-1:
• Human Gzm B consensus: LIGADVLVQ 6.5ul substrate in 38.5ul PBS with
5ul MCP-1.
• Rat Gzm B consensus: LIETDSGL 5ul substrate in 40ul PBS with 5ul MCP-1.
• Mouse Gzm B Consensus: LIGFDVGVQ 7ul substrate in 38ul PBS with 5ul
MCP-1.
Chromogenic Substrate Assay
A battery of chromogenic substrates is also used to see if there is any other
activity not specifically restricted to tryptase. These substrates have a chromophore called
para-nitroaniline when cleaved of emits light. The spectrophotometer is set at 405nm
wavelength because of low background reading contamination with substrate. In a clear
96 well plate that is used to set blank for photometer add 195ul of PBS and 5ul of 8mM
substrate; one well for each substrate. Then allocate wells for all substrates for, in this
case 2 enzymes; chicken CTSG & alligator MCP-1. Each experimental well will contain
190ul of PBS, 5ul of 8mM substrate and 5ul of enzyme. Substrates ordered from Sigma
Aldrich.
The substrates are as follows:
• Suc-Ala-Ala-Pro-Phe-pNA Chymase Substrate
• Suc-Leu-Leu-Val-Tyr-pNA Chymase Substrate
• Suc-Ala-Ala-Pro-Ala-pNA Elastase Substrate
• Suc-Ala-Ala-Pro-Ala-pNA Elastase Substrate

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• Suc-Ala-Ala-Pro-Leu-pNA Elastase Substrate
• Suc-Ala-Ala-Pro-Val-pNA Elastase Substrate
• Boc-Val-Leu-Gly-Arg-pNA Tryptase Substrate
• Z-Gly-Pro-Arg-pNA Tryptase Substrate
• Ac-Tyr-Val-Ala-Asp-pNA Aspase Substrate
• Ac-Val-Glu-Ile-Asp-pNA Aspase Substrate
The plate is then measured every 10 minutes for the first 2hr, every 30 minutes for the 3rd
hour, every 1hr for hours 4-6 and at 24hr, between these readings, reaction plate (96
wells) is left in the 37°C room.
Phage Display
Figure 8. A Schematic drawing of the Phage Display procedure
(Adopted from Ulrika Karlsson. Cutting Edge- Cleavage Specificity and Biochemical
Characterization of Mast Cell Serine Proteases. Acta Universitatis Upsaliensis. Uppsala 2003.)
(Studier et al for more T7 bacteriophage information) (16)
A phage library containing 5x107
of T7 bacteriophages with one of the coat
proteins containing an extra 9 amino acids long randomized region and a histadine tag
which is used to adhere to the positive nickel beads was used to try to determine the
extended specificity of these two enzymes. Incubation of the phage library with the nickel

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beads lasts one hour at +4 o
C allowing phages to bind. Ten wash steps for the first day
and fifteen for days 2-5 with PBS tween 0.05%, 1M NaCl was then used to remove all
unbound phages. After washing, the nickel beads (Ni-NTA) were resuspended in 375ul of
PBS and the specific protease was added; the amount of enzyme added depends not only
on the concentration, but primarily the activity. This amount to be used was deduced
from the SDS-PAGE gel assays after expression and purification.
Once the enzyme is added to the beads with phages bound it is incubated at 37o
C
for 2 hours in parallell with a PBS control (same binding/washing steps with no enzyme).
During this stage, a culture of the E.coli BLT 5615 is inocluated in 100ml of LB Amp
media. BLT 5615 bacteria have a specific T7 promoter region responsible for increasing
production of phage coat protein when fed to the phages for amplification. Preparation of
top agar for plating is done by adding 0.9g of agarose to 150ml of LB media, heating to
boil then keeping in hot water bath at 55°C until plating.
After 2 hours of incubation with protease and preparation of a serial dilution set
for both the enzyme and the PBS control. 30ul of supernatant is taken after centrifugation
at 4G and added to 270ul of LB Amp to make a 10-1
dilution; this is continued to a series
of dilutions to 10-6
for both samples. The remaining amount of supernatant is transferred
to an Eppendorf tube containing 30ul of Ni-NTA beads and 100ul PBS for amplification
of the phage later. The tubes now only containing used Ni-NTA beads are mixed with
100ul of 100mM imidazole to release all bound phages which a 100ul of solution, after
vortex/centrifuge are added to 900ul of LB Amp for dilution up to 10-6
as well.
The next step is plating; where, depending on the dilution series you will add
100ul of IPTG to each 14ml round-bottom falcon tube followed by 100ul of your
appropriate dilution. When OD 600nm reaches 0.5 of the BLT bacterial culture, 10ml of
the bacteria solution is added to two different 50ml Falcon tubes and 100ul of IPTG is
added. These two tubes are incubated for 30min at 37°C. After 30 minutes of incubation
with IPTG, add rest of cleaved phage from earlier and incubate at 37C for 75min.
BLT bacteria are then added to each of the round-bottom tubes at 300ul
increments. Then when appropriately labeled plates are warm and the top agarose is at
55°C plating can begin. Pipetting of 3ml of top agarose into the IPTG/Phage dilution/BLT
mixture swirl and pour onto the corresponding plate, making sure the top agarose is

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evenly distributed. Continue this for dilution sets (usually 10-4
-10-7
) depending on
previous days, then store plates at 37°C for 2 hours 30 minutes.
The OD should eventual reduce because of amplification of cleaved phages. The
top agarose allows for bacterial growth but bacteria infected with phage will burst
resulting in a plaque. Counting of plaques allows quantification of phage cleavage by our
specific enzyme. Plates and amplified phage are then stored at +4°C. 1.5ml of amplified
phage is centrifuged at 4G for 3 min; 800ul of supernatant is then added to 100ul of 5M
NaCl and 100ul of PBS for use the next day.
After 5 days of biopanning (selecting for the cleaved phage phenotype) you
should see a large difference between plaques produced on enzyme plates at the same
dilutions as there are for the PBS control. Pick these plaques with glass Pasteur pipets and
shake them for 30min in 100ul phage lysis buffer; 100mM NaCl, 20mM Tris, 6mM
MgSO4 in dH2O. 1ul of this is then added to PCR tubes with 49ul of master mix
consisting of 5ul of taq 10x buffer with MgCl2, 1ul of 5pmol/ul of both 5’ & 3’ T7 primer,
1ul dNTP mix 10mM, 0.5ul taq polymerase and 40.5ul dH2O.
PCR Conditions:
40 Cycles
94°C 5min for initial denaturation
94°C 50secs for denaturation
50°C 60secs for annealing
72°C 60secs for extension
72°C 6min for final extension
Hold at +4°C
PCR results are then run on DNA acrylamide gel electrophoresis to make sure
PCR fragment is present. These fragments are loaded into a 96 well plate and then sent to
GATC in Germany for Sanger sequencing.
T7 5’ forward primer for sequencing: GTTAAGCTGCGTGACTTGGCT.
If sequences come back showing a definitive pattern there is an arrangement process,
along with a statistical analysis of amino acids in P5-P5’ positions, alluding to the natural
affinity a protease has towards specific substrates.

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Results
Based on the evolutionary analysis described in the introduction section two
proteases were selected for further analysis, chicken CTSG and Chinese alligator MCP-1.
The coding sequences for these two proteases were compiled and ordered as designer
genes from Genscript Corporation. Following arrival of the clones it is important to
establish that the vectors are indeed holding the required sequences of each protease. This
was done through transformation and multiplication of DK1 E.coli. The restriction
enzyme cleavage of the vector and subsequent run on DNA acrylamide gel
electrophoresis shows that the inserts are present and ready to be transfected with vector
into our HEK 293 mammalian expression model. Most of the sequences are around 750
base pairs in length; further indicating gene homology and structural similarities. This is
the first step in assuring proper expression and an assessment of sample purity. Alligator
MCP-1 and Chicken CTSG are the enzymes that will be studied by mammalian cell line
expression, quantification and analysis in the scope of this report.

Figure 9. Protease Insert & Vector Restriction Enzyme cleavage
Restriction enzyme cleavage of the vector using EcoRI and XhoI, of which sites border the insert for easy
manipulation. Top bands belong to the pCEP-pu2 vector while the bands of around 750bp in size are the
inserts coding for the different proteases. There are a total of 22 proteases in the process of being
categorized, this being the first set. Insert is found around 750bp while the vector is up around 10kb.

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The next step is the successful transfection and expression in HEK 293
mammalian cell lines, which requires growth and a puromycin selection process. Once
the selection process has removed all but the cells that have the selected episomal vector
its all about growth and expansion. As the DMEM media starts to change from a red to
yellow harvesting and purification via Ni-NTA beads yields varying quantities of either
chicken CTSG or Alligator MCP-1. SDS-PAGE shows positive for high concentrations
of both proteins.

Figure 10. Elution fraction of Chicken CTSG and Alligator MCP-1
Elutions of different harvests from 175cm2
flasks of both chicken and alligator show higher concentrations
in chicken but relatively good purity for both. These concentrations are both viable for specificity
experiments, assuming activity is present. Each elution set is done with 500uM imidazole and the 1st
fraction is omitted, as it normally has no protein.

Once the presence of the desired proteins has been established (Fig. 10) its
necessary to quantify the amount with the Bradford Assay (Fig 11.). This is done using
the chemical change between the red to blue forms of Coomassie dye via electron
donation and chelation with the supplied protein. Measured by the spectrophotometer at
405nm, the absorbance is then put through an exponential equation to give estimated
concentration. This is important to measure the activity of the enzyme after cleavage
assays are done, also to estimate the amount of enzyme needed for the assays themselves.
The results from the Bradford assay are only an estimate because of the likelihood of
other proteins in the sample, which will increase the values received by the
spectrophotometer (Fig. 11). These values are more than enough to work with since about

19
5ml was harvested for Chicken CTSG and 2ml was harvested for Alligator MCP-1 in
these concentrations.
Chicken CTSG High Elution
Dilution Factor
(DF)
X-values
(OD)
Y-Values
equation Y-values x DF
Average
Concentration
5x 0.04 0.1095 0.5475
2x 0.092 0.2703 0.5406 0.5ug/ul
1x 0.153 0.5459 0.5459
Low Elution
5x 0.019 0.0534 0.267
2x 0.046 0.1261 0.2522 0.2ug/ul
1x 0.07 0.1968 0.1968
Alligator MCP-1
5x 0.025 0.0699 0.3495
2x 0.06 0.1663 0.3326 0.3ug/ul
1x 0.072 0.203 0.203
Figure 11. Bradford Assay Chicken CTSG & Alligator MCP-1
The concentrations are based on spectrophotometer readings then put into the equation (y=50.724x3
-
1.8662x2
+2.6431x+0.0035) to provide an estimate of protein concentration based on the interaction with
the coomassie blue dye. Two concentrations of CTSG were measured 0.5ug/ul for high and 0.2ug/ul for low.
MCP-1 was 0.3ug/ul.

Usually the elutions also contain bovine serum albumin, which comes from the
media the cells are grown in and attaches itself to the beads as well. The streaking seen in
some SDS-PAGE are partially digested proteins of various sizes that are dragged along
with the protein of interest creating a blurry impure sample image. If absolute purity was
a necessity or if other active compounds could be contaminating the sample,
repurification with Ni-NTA beads is a possible solution. Instead, comparing the Bradford
assay with a diluted bovine serum albumin series would give a better idea of
concentration seen on gel rather than OD.
Since the general concentration range for the two proteases had been established
(Figs. 10,11), the next step was for activation of each 100ul aliquot enzyme with 1.5-
2.0ul of enterokinase. This process theoretically mimics the maturation of the protease
through natural channels. Zymogen maturation occurs with the cleavage of a small
peptide covering the active site of the enzyme, allowing for interaction with substrates.

20

Figure 12. Bovine Serum Albumin Concentration Series
Bovine serum albumin was taken from stock and made into specific dilutions. These are then compared to
newly expressed and harvested proteins to compare concentrations on gel. From the SDS-PAGE seen here,
Chicken CTSG has a concentration 1st
of 0.4ug/ul then 0.8ug/ul, while Alligator MCP-1 has a
concentration of 0.1ug/ul-0.2ug/ul.
Since the enterokinase site is only 5 amino acids long and the area cleaved away is 14-16
amino acids in length it is sometimes hard to tell if high concentrations of enzyme have
been activated. This is remedied by running smaller concentrations after exposure to
enterokinase to ensure a difference in size can be seen on gel. It’s also important to run
the SDS-PAGE as long as possible to get the separation necessary for discerning the
difference between the pieces.
Chicken CTSG and Alligator MCP-1 have been activated as seen in Fig. 13.
Moving forward from here with CTSG is straightforward because the triplet is already
known as aspartic acid in the 189th
position with two glycines in the 216th
and 226th
positions, showing a tryptase triplet with preference to basic amino acids like arginine in
the P1 position. Alligator MCP-1 although, is still a mystery. The first and one of the
easier experiments that could be done was the recombinant substrate assay with
previously ordered substrates for previous proteases. A set of substrates used in the
catfish study done by Michael Thrope in 2014 were still held in -70°C freezer and were
used as a first attempt to study the specificity of these two proteases.

21

The first two substrates that were tested (Fig 14.) had multiple arginines in and
around the P1 position, which should be high priority targets for chicken CTSG having a
negative amino acid in the 189th
position, or the S1 pocket. While there was what looks to
be cleavage of each of the substrates, this never increases from the 0 min time point,
indicating no rise from starting point and no specific protease cleavage of these two
substrates. Another catfish substrate variant that showed met-ase activity was also
available, which was run as a recombinant substrate assay as catfish V1 (Fig 15.)
The overall goal for these two proteases is to understand their specificity,
so in essence, taking clues from any and all directions is important to moving towards a
conclusion. Since no conclusive cleavage occurred with tryptase substrates its time to try
something else. Catfish V1 substrate with the sequence RVTGMSLV presents as a met-
ase substrate because of the methionine in the P1 position.
From figure 15 no cleavage is seen, indicating no enzyme preference for
methionine which could be predicted for CTSG but needed clarification for MCP-1
because of the proximity of both proteases to the met-ase branches on the phylogenetic
tree (Fig. 3).
Enterokinase cleavage of
Chicken CTSG and Alligator
MCP-1
-EK +EK
Chicken
-EK +EK
Alligator
Figure 13.
Enterokinase
Cleavage of Chicken
CTSG and Alligator
MCP-1
Seen on SDS-PAGE
Chicken CTSG and
Alligator MCP-1 are
presented un-cleaved
and cleaved. The
second column in each
case is 14-16 amino
acids shorter showing
the protein has been
activated after 2hr
incubation in 37°C
with enterokinase.
After protein has been
activated it can be
used in cleavage
specificity assays.

22

Figure 14. Recombinant Substrate Assay with Catfish V2-3
CTSG & MCP-1 were run on recombinant substrate assay. The time points were 0 min, 15 min, 45 min &
150 min, which is the time after the enzyme was added the the master mix (containing substrate at 0.5ug/ul
with remaining PBS). Catfish V2 substrate has the sequence VVRRAAAG while V3 has VVRRRAAG to test
tryptase-like proteases. No conclusive cleavage was observed for either substrate.

As a diagnostic check, because of the ambiguity of the alligator MCP-1, another battery
of recombinant substrates were attempted. These are all chymase substrates having larger
aromatic amino acids in their P1 position. This was only attempted for alligator MCP-1
because the triplet for chicken CTSG is already known. These substrates come from
previous studies done with other appropriate labeled proteases: Human chymase,
Opossum chymase and a variation of Human chymase 6. Human chymase sequence
VVLFSEVL has a phenylalanine in the P1 position while the opossum chymase with
sequence VGLWLDRV contains a tryptophan, another aromatic amino acid. The third
and less notable substrate was a variation on human chymase consensus with a sequence
of VVLLSEVL with a leucine in the P1 position which chymases have been shown
historically to favor as well (8).
Two more attempts at recombinant substrate assays were performed. Chicken
CTSG was run with an elastase V1 substrate (SGRGGRGGRGV) with no visible
cleavage (gel not shown) and Alligator MCP-1 was run with three granzyme B
substrates: human, rat and mouse, with sequences LIGADVLVQ, LIETDSGL and
LIGFDVGVQ respectively with no cleavage (gel not shown).

23

Figure 15. Catfish V1 Recombinant Substrate Assay
A recombinant substrate assay for both CTSG & MCP-1 with a met-ase substrate catfish V1: with a
sequence of RVTGMSLV. Similar time points are used in all recombinantassays: 0 min, 15 min, 45 min &
150 min. No cleavage from either enzyme for this substrate occurred.

Figure 16. Recombinant Substrate Assay For Alligator MCP-1
This recombinant substrate assay was specifically done for Chinese alligator MCP-1. Similar time points
were used: 0min, 15min45min & 150min with each substrate being 0.5ug/ul in the master mix
before protease was added. No cleavage occurred with any of the chymase substrates illustrating no
favorable interaction with MCP-1.

24
With the results from each of the quintessential recombinant tryptase, met-ase,
chymase, elastase and asp-ase substrates coming up negative, another assortment of
substrates could be attempted with a slightly different methodology. Chromogenic
substrates enlist a chromophore right after a short peptide ending with the hopeful P1
position amino acid. Over a specified time period the cleavage of these substrates will
result in increasing chromophore release measured by a plate reading spectrophotometer
(Fig. 17). These results also proved unhelpful, giving almost zero signal over a 24 hour
period with the exception of two tryptase substrates. This cleavage signal however is
most likely contributed by the enterokinase that was added to activate our proteases.
Unfortunately, no clues have presented themselves in order to narrow the search
for the perfect substrate for either enzyme. This paired with the recombinant substrate
assay’s inconclusive results forced a necessary expansion of protocol, leading to the
introduction of the phage display assay. This technique can be decisive because of its
broad library of substrates to choose from; however is limited in the respect of
biopanning and post-production work taking up to two weeks before results are
interpreted, along with the tendency for ultra specific proteases to be left behind due to
very few perfect substrates may be present in the library. This protocol is where the
majority of time was spent in an attempt to determine the pattern of substrate selection
for both proteases. The results from phage display were not immediately forthcoming
due to struggles with phage contamination and the variability that was seen in the results.
What is shown in figure 18 is the culmination of months of phage display modification,
optimization and in essence, experimentation with a protocol that was already established
as a working model. The trend of CTSG and MCP-1 shows the expected growth phase
from day 2 to day 3 or 4 but in all cases never continues (Fig. 18). The selection process
falls off and the difference between CTSG/MCP-1 and PBS diminishes. The trend that
was observed is usually a steep drop in selectivity from 10-15x PBS plaque-forming units
(PFU) to equal or 2x-3x PBS control, indicating a loss off specific phage. The graphs
represent the ratios between the PBS control and the protease PFU in question. The ratios
themselves are created from the averaging of the PFU for each day and dividing them by
PBS values.

25

Figure 17. Chromogenic Substrate Results for CTSG & MCP-1
Chromogenic substrate reactions over a 24-hour period show no cleavage except for two tryptase
substrates towards the last time point for both enzymes. This most likely represents the enterokinase that
was used for enzyme activation instead of the enzymes themselves. No conclusive enzymatic cleavage.

26

Figure 18. Phage Display Ratio Representation of CTSG & MCP-1 vs. PBS
CTSG & MCP-1 ratios vs. PBS control show average PFU for each day. Averaging the PFU and then
dividing CTSG or MCP-1 values by PBS, then setting PBS to 1 calculate ratios for the graphs. All three
graphs indicate selection increase from day 2 to day 4 or 5 and then a decrease.
0.00
5.00
10.00
15.00
20.00
25.00
30.00
1 2 3 4 5
Ratio
Day
CTSG & MCP-1 vs. PBS 29/3/16
CTSG
MCP-1
PBS
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
1 2 3 4 5
Ratio
Day
CTSG & MCP-1 vs. PBS ratio 18/4/16
CTSG
MCP-1
PBS
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
1 2 3 4 5 6
Ratio
Day
CTSG & MCP-1 vs. PBS Ratio 2/5/16
CTSG
MCP-1
PBS

27
The plaques were counted each day for each dilution of the series and multiplied
by the dilution factor giving how many plaque-forming units (PFU). These were then
averaged per day for each protease and divided by the PBS average PFU to give a ratio.
The selection rounds where the highest difference between the protease PFU and PBS
control were selected, plaques were gathered amplified by PCR and sent for sequencing.
The sequences revealed were a mixed assortment of potential substrates but were
submerged in to many background phages with identical sequences that are continually
present during many previous phage display attempts. Three separate sets of 96 samples
(plaques) were sent for sequencing with similar results. Unfortunately without further
selection i.e. the differences between protease and PBS PFU, there are no identifiable
patterns emerging from the data.
Discussion
The chain link relationships that follow these proteases through hundreds of
million years of evolution into some of the most versatile biological compounds ever
studied. The push and pull of random mutation forced to heel by ruthless environmental
pressure ushers in a particularly efficient set of attributes for any organism to further
optimize its genome. The most difficult task is to try and decipher the seemingly random
changes seen in these genomes, the seemingly random manipulations of attribute and
function to fuse a semblance of an idea together. The project itself belies a simple
expression, quantification procedural method; this however is a gross over simplification.
Each step along the way could hide pitfalls threatening a positive, conclusive result and
this discussion is an attempt to relay and move forward.
From the early stages of this project the purpose was to express and quantify a
useable amount of each of the proteases through the transfection into the HEK 293 cell
line. This was achieved and exceeded expectations, gathering a number of properly
folded proteases for studies of their primary and extended cleavage specificities to further
aid the desciphering of their appearance and diversification during vertebrate evolution.
With the expression of these enzymes being seen on the gel, it does not necessarily mean
they are functional. They have been isolated and purified so the HIS tags are visible to
the NI-NTA beads providing evidence that at least that part of the protein has folded

28
correctly. Previous literature has provided foundation for HEK 293 episomal expression
being capable of producing viable proteases in their correct conformations (17,18). This
however, may not translate to every protein, some previous work with neutrophil elastase
encountered harvesting problems due to toxicity of the mutation expressed. The unfolded
protein response is a kind of check and balance system within the cell to make sure
translation, modification and transport of newly created proteins goes as planned or is
degraded and removed from the assembly line (19). While the HEK 293 cells grew well
and were not overly apoptotic, foreign proteases could theoretically build up between the
ER and Golgi apparatus and be released giving a false positive for functionality. The
availability of the enterokinase site paired with the witnessed above base-line cleavage
seen in the tryptase recombinant substrate assays (Fig. 14) and phage display models (Fig.
18) however, shows a different possibility.
Even if the produced proteases are properly folded they must be activated first to
be able to cleave its potential substrates. The enterokinase sites used in this model are
used because of its very high specificity (20). The maturation process of proteases in-vivo
occurs through cleavage of an inactive zymogen. After being cleaved (activated) and
transported to granules or activated extracellularly as for example prothrombin (21). This
system is mimicked by the enterokinase activation system and is in place to reduce
erroneous cleavage within the cell. However the enterokinase, stays in the solution
containing the protease and while it is specific in its cleavage is thought to be responsible
for some of the low response cleavage seen in the chromogenic assay. Having the
enterokinase in the solution seems to be a necessary evil because with activation cleavage
the His tag on the protease is removed and subsequent purification becomes extremely
difficult, even though small substrate interaction with enterokinase still exists. Once
activated the enzymes are ready for cleavage specificity experimentation, which should at
least give hints as to protease function.
Recombinant substrate assays are easily done with materials that are already on
hand. Trying the pre-existing substrates that represent the main classifications of serine
protease was the best approach to establishing cleavage specificity for both Chicken
CTSG and Alligator MCP-1. Unfortunately all of the recombinant substrates proved
unfavorable for these two proteases probably due to a very high extended specificity of

29
these two proteases. In figure 14 there are bands on the gel below the uncleaved
substrates indicating either an impurity in the substrate solution or a cleaved portion of
the substrate. The results are inconclusive because these bands are present throughout all
the time points and neither increase or decrease, but could also be contributed to the
presence of enterokinase. This resistance towards cleavage of these substrates lends to
the idea that both of these proteases are extremely specific, requiring multiple
interactions outside the catalytic triad and triplet for cleavage to occur (22).
Chromogenic substrate assays are also able to provide limited data towards
cleavage specificity because of the attachment of the chromophore to the P1 position
amino acid, allowing for some upstream selectivity with P2-P5. This however; further
limits a favorable interaction between protease and substrates because of immediate
downstream interactions aren’t available with P1’-P5’ position amino acids (7). In order
to tackle the problem of specificity from a different angle we decided to provide the
proteases with more possible targets.
Phage display increases the chance of finding a substrate drastically, providing
5x107
randomized nonamer phage clones available for proteolytic cleavage (23). While
the sheer amounts of phage combinations make it possible to select from a larger library,
positive results were still not achieved. A general trend that appeared (Fig. 18) was 2-3
days of increased selection versus the PBS control then a decline in plaques. This resulted
in untraceable patterns of sequence reports accompanied by 40-70% background phage,
indicating little to no selection had occurred. The phage display protocol involves many
steps that could influence, the result. These were tested to make sure the previously
successful protocol was still viable, with emphasis on HIS tag availability for phage
capture and overall library variability.
Each day of phage display protocol is almost identical with slight variations of the
dilution series based on previous plaque counts so accurate comparisons can be made
between proteases and PBS. An important step that occurs each day is the incubation of
E.coli BLT5615 with IPTG. This step is important for the generation of coat protein for
the bacteriophages, however this coat protein is unmodified, containing no HIS tags or
nonamer sequences to be cleaved. Alteration of the timing of incubation with IPTG could
theoretically increase or decrease the amount of HIS tagged coat proteins available on the

30
coat surface, essentially affecting the binding to the Ni-NTA beads. The protocol states
that 30 minutes of IPTG incubation for 10 ml of 0.5OD BLT5615 culture is sufficient for
an appropriate ratio of non-modified coat protein to HIS tagged. This ratio however could
be adjusted depending on protease activity and specificity to try and optimize cleavage
environment. CTSG and MCP-1 may be extremely specific, and possibly so because of
varied interaction sites outside the immediate P5-P5’ within the S pocket (21). An
increase in induction time with IPTG would, when incubated with phage, reduce the
amount of HIS tagged coat proteins, possibly reducing steric hindrance for the protease
and allowing nonamers to be more readily found and cleaved.
The T7 phage library has 5x107
nonamer variations, however after receiving
sequencing of results of the highest selection rounds with both proteases, there was a
large contingent of background phage: identical nonamer sequences found throughout
multiple biopanning attempts with different enzymes. This, paired with a large
discrepancy between elution plaque counts (using imidazole to flush remaining phages
from beads after dilutions) from day 1 to day 2 signified a limited pool of phage, possibly
reduced by storage or overpopulation of background during incubation and creation.
Represented in the 3rd
graph in figure 18 is an attempt at expanding the library (induction
of BLT 5615 with IPTG and incubation of phage library with 2x the Ni-NTA bead count
and 5x the phage library volume) to saturate the beads and provide as many nonamer
variations for cleavage as possible. Unfortunately, as seen in figure 18 the selection
process again stalled towards day 5, this method however, could be continued and with
some good fortune provide conclusive results.
The goal of this study is provide experimental evidence of the bioinformatic
relationships established in Akula et al. 2015 (8), and there is plenty of work to do with a
library of expressed and quantified proteases from over 25 different species. With each
protease however, comes its own set of challenges. The activity varies, the cleavage
specificity may not reflect the dogma and alterations of the loci and phylogenetic trees
are changing on a daily basis with complete organism genomes being sequenced almost
as frequently. With optimization of the current techniques in the lab with and increase in
substrate libraries, progress and data will not be hard come by, adding even more
complexity to the evolutionary story. The failure to pinpoint any conclusive substrates

31
for these proteases only leads the search elsewhere, the work done was not in vain, just
indicative of necessary manipulation of the methods.
“Science is the systematic classification of experience” –George H. Lewes

Acknowledgements
Professor Lars Hellman, for his patience, guidance and overwhelming knowledge base
providing invaluable input in all things immunity (among many other amazing anecdotes).
PhD Srinivas Akula, for his friendly demeanor, helpful attitude and willingness to teach
and be taught. Also he taught me everything I know about cricket.
Post-doc Zhirong Fu, for her appreciative perception of the world, dedication to her work
and showing me all the things I could probably do better.
Master Student Payal Banerjee, for sharing the office with a basket case and tolerating
my rambling during the tough times and thesis work, I’m not sure I could’ve done it
without you.
PhD Gurdeep Chahal, for showing an ignorant 1st
year master student the ropes and
dealing with my seemingly endless stream of questions.
The entire A8: 2-laboratory crew for showing me nothing but support and guidance and
tolerating a loud American for the duration.

32
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