Serine Proteases, A Basis of ImmunityThrough Evolution

Luke Morton

Degree project in biology, Master of science (2 years), 2016Examensarbete i biologi 45 hp till masterexamen, 2016Biology Education CentreSupervisor: Lars HellmanExternal 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 oC 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 37oC

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

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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-1vs.PBS29/3/16

CTSGMCP-1PBS

0.001.002.003.004.005.006.007.008.009.0010.00

1 2 3 4 5

Ratio

Day

CTSG&MCP-1vs.PBSratio18/4/16

CTSGMCP-1PBS

0.002.004.006.008.0010.0012.0014.0016.00

1 2 3 4 5 6

Ratio

Day

CTSG&MCP-1vs.PBSRatio2/5/16

CTSGMCP-1PBS

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

References

1. Wouters MA, Liu k, Riek P, and Husain A. 2003. A Despecialization Step Underlying Evolution of a Family of Serine Proteases. Molecular Cell 12.2: 343-54. 2. Stroud RM. 1974. A Family of Protein-Cutting Proteins. Scientific American 231.1: 74-88. 3. Rawlings ND, Barrett AJ. 1994. Families of Serine Peptidases. Methods in Enzymology Proteolytic Enzymes: Serine and Cysteine Peptidases 19-61. 4. Dores RM, Rubin DA, Quinn TW. 1996. Is It Possible to Construct Phylogenetic Trees Using Polypeptide Hormone Sequences? General and Comparative Endocrinology 103.1: 1-12. 5. Sundström G, Larsson TA, Brenner S, Venkatesh B, Larhammar D. 2008. Evolution of the Neuropeptide Y Family: New Genes by Chromosome Duplications in Early Vertebrates and in Teleost Fishes. General and Comparative Endocrinology 155.3: 705-16. 6. Caughey G. 2006. A Pulmonary Perspective on GASPIDs: Granule-Associated Serine Peptidases of Immune Defense. CRMR Current Respiratory Medicine Reviews 2.3: 263-77. 7. Hellman L, Thorpe M. 2014. Granule Proteases of Hematopoietic Cells, a Family of Versatile Inflammatory Mediators – an Update on Their Cleavage Specificity, in Vivo Substrates, and Evolution. Biological Chemistry 395.1 8. Akula S, Thorpe M, Boinapally V, Hellman L. 2015. Granule Associated Serine Proteases of Hematopoietic Cells – An Analysis of Their Appearance and Diversification during Vertebrate Evolution. PLOS ONE 10.11 9. Pham CTN. 2006. Neutrophil Serine Proteases: Specific Regulators of Inflammation. Nature Reviews Immunology 6.7: 541-50. 10. Korkmaz B, Moreau T, Gauthier F. 2008. Neutrophil Elastase, Proteinase 3 and Cathepsin G: Physicochemical Properties, Activity and Physiopathological Functions. Biochimie 90.2: 227-42. 11. Hedstrom L. 2003. Serine Protease Mechanism and Specificity. ChemInform 34.6 12. Birktoft JJ, Blow DM, Henderson R, and Steitz TA. 1970. The Structure of Alpha-Chymotrypsin. Philosophical Transactions of the Royal Society B: Biological Sciences 257.813: 67-76.

33

13. Kohfeldt E, Maurer P, Vannahme C, Timpl R. 1997. Properties of the Extracellular Calcium Binding Module of the Proteoglycan Testican. FEBS Letters 414.3: 557-61. 14. Inada M, Izawa G, Kobayashi W, Ozawa M. 2016. 293 Cells Express Both Epithelial as Well as Mesenchymal Cell Adhesion Molecules. International Journal of Molecular Medicine. 15. Thomas P, Smart TG. 2005. HEK293 Cell Line: A Vehicle for the Expression of Recombinant Proteins. Journal of Pharmacological and Toxicological Methods 51.3: 187-200 16. Studier WF, Moffatt BA.1986. Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes. Journal of Molecular Biology 189.1: 113-30. 17. Andersson MK, Enoksson M, Gallwitz M, Hellman L. 2008. The Extended Substrate Specificity of the Human Mast Cell Chymase Reveals a Serine Protease with Well-defined Substrate Recognition Profile. International Immunology 21.1: 95-104. 18. Thorpe M. 2012. Catfish Protease Extended Cleavage Specificity Unpublished Data. 19. Nustede R, Klimiankou M, Klimenkova O, Kuznetsova I, Zeidler C, Welte K, Skokowa J. 2015. ELANE Mutant-specific Activation of Different UPR Pathways in Congenital Neutropenia. British Journal of Haematology 172.2: 219-27. 20. Terpe K. 2003. Overview of Tag Protein Fusions: From Molecular and Biochemical Fundamentals to Commercial Systems. Applied Microbiology and Biotechnology 60.5: 523-33. 21. Bradford HN, Krishnaswamy S. 2016. The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase. Journal of Biological Chemistry. 22. Graf L, Craik CS, Patthy A, Roczniak S, Fletterick RJ, Rutter WJ. 1987. Selective Alteration of Substrate Specificity by Replacement of Aspartic Acid-189 with Lysine in the Binding Pocket of Trypsin. Biochemistry 26.9: 2616-623. 23. Thorpe M, Yu J, Boinapally V, Ahooghalandari P, Kervinen J, Garavilla LD, Hellman L. 2012. Extended Cleavage Specificity of the Mast Cell Chymase from the Crab-eating Macaque (Macaca Fascicularis): An Interesting Animal Model for the Analysis of the Function of the Human Mast Cell Chymase. International Immunology 24.12: 771-82.