University of Southern California Dissertations and Theses

Controlled ocular drug delivery using peptide-mediated phase separation

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Controlled ocular drug delivery using peptide-mediated phase separation

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CONTROLLED OCULAR DRUG DELIVERY USING PEPTIDE-
MEDIATED PHASE SEPARATION

by

Aarti Jashnani

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

May 2010

Copyright 2010 Aarti Jashnani

ii

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Andrew Mackay for his guidance, enlightenment
and encouragement throughout the project. I would also like to show my appreciation
towards my thesis committee members Dr. Sarah Hamm-Alvarez who has collaborated
with this project and also Dr.Curtis T.Okamoto for their guidance, advice and time. A
special thanks and appreciation to Pang-Yu Hsueh from Dr. Hamm-Alvarez’s laboratory
for providing me with the Valine library and Francie Yarber from Dr. Hamm-Alvarez’s
laboratory for providing the rabbit tears. I greatly appreciate all the help and guidance
from my colleagues in the Mackay Laboratory in particular Erick Medina , Ara Moses,
Guoyong Sun, Martha Pastuszka, Mihir Kunjeshkumar Shah, Sejal Parakh, Siti Mohd
Janib, Suhaas Aluri, Vinod Valluripalli, Wan Wang. I would also like to thank my family
and friends for their encouragement and support throughout my master’s program.
Finally, I would like to show my gratitude and appreciation to the USC School of
Pharmacy for supporting this project.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS viii
ABSTRACT x
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: BACKGROUND 3
2.1 The tear film 3
2.2 Dry eye syndrome 4
2.3 Elastin-like polypeptides 6
2.3.1 Recombinant Synthesis of the ELP library 8
2.3.2 Purification of ELPs using Inverse transition cycling 10
2.4 Lacritin 11
2.4.1 Structural attributes of lacritin 12
2.4.2 Implication in various diseases 14
2.5 Synthesis of lacritin-ELP fusion protein 15
2.6 Specific Aims 16

CHAPTER 3: MATERIALS AND METHODS 18
3.1 Materials 18
3.2 Design of Lacritin-ELP using Laser gene 18
3.3 Recombinant synthesis of Lacritin into pET25b+ vector 21
iv

3.4 Recombinant synthesis of V
96
into pET25b+ vector containing Lacritin 22
3.5 Protein purification using ITC 23
3.6 Beckman Coulter DU 800 UV-vis spectrophotometer 25
3.7 SDS-PAGE 26
3.8 Degradation studies 26
3.9 Thrombin cleavage assay 27

CHAPTER 4: RESULTS 28
4.1 Characterization of the Valine Library using UV-vis spectrophotometer 28
4.2 Confirmation of the purity and molecular weight of the ELPs 35
4.3 Degradation of ELPs in physiological fluids 36
4.4 Comparison of T
t
for V
96
in PBS and tears 37
4.5 Confirmation of lacritin and fusion protein sequence in pET25b+ 38
4.6 Characterization of lacritin-V
96
using UV-vis spectrophotometer 40
4.7 SDS-PAGE of fusion protein 42
4.8 Thrombin cleavage assay 44

CHAPTER 5: DISCUSSION AND CONCLUSION 46

CHAPTER 6: FUTURE DIRECTIONS 48

REFERENCES 50

v

LIST OF TABLES

Table 1: Construct of ELP and Lacritin-ELP 19
Table 2: Summary of the predicted T
t
for the valine library 33
Table 3: Summary of the experimental T
t
for the valine library 34
Table 4: ELP constructs along with their predicted ocular clearance 35
Table 5: Summary of the T
t
for the fusion protein 41

vi

LIST OF FIGURES

Figure 1: Phase transition of ELPs 7
Figure 2: Steps involved in RDL 9
Figure 3: Inverse transition cycling (ITC) 10
Figure 4: Binding of Lacritin onto syndecan-1 14
Figure 5: Synthesis of Lacritin-ELP fusion protein 16
Figure 6: Turbidity plot for ELP, G (VPGVG)
96
Y 17
Figure 7: Lacritin sequence 20
Figure 8: Log plot of the turbidity of different ELP
chain lengths at various concentrations 29
Figure 9: Turbidity plot for ELP, G (VPGVG)
24
Y 30
Figure 10: Turbidity plot for ELP, G (VPGVG)
36
Y 30
Figure 11: Turbidity plot for ELP, G(VPGVG)
48
Y 31
Figure 12: Turbidity plot for ELP, G(VPGVG)
144
Y 31
Figure 13: Turbidity plot for ELP, G(VPGVG)
192
Y 32
Figure 14: SDS-PAGE of valine library proteins 36
vii

Figure 15: SDS-PAGE of ELPs incubated in tears 37
Figure 16: Concentration dependence of T
t
in physiological solutions 38
Figure 17: Agarose gel for diagnostic digest of lacritin 39
Figure 18: Agarose gel for diagnostic digest of Lacritin-V
96
40
Figure 19: Turbidity plot of Lacritin-V
96
41
Figure 20: SDS-PAGE of fusion protein with coomassie staining 43
Figure 21: SDS-PAGE of fusion protein with copper chloride staining 44
Figure 22: Cleavage of fusion protein using thrombin 45

viii

ABBREVIATIONS

CIP Alkaline phosphatase, Calf intestine
DES Dry eye syndrome
DMSO Dimethyl sulfoxide (DMSO)
E. coli Escherichia coli
ELP Elastin-like polypeptides
ITC Inverse transition cycling
Lac-V
96
Lacritin and G (VPGVG)
96
Y fusion protein
M.W. Molecular weight
RDL Recursive directional ligation
PBS Phosphate buffer saline
PEI Poly (ethyleneimine)
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
TB Terrific broth
T
t
Transition temperature
ix

V
24
G (VPGVG)Y
24
V
36
G (VPGVG)Y
36

V
48
G (VPGVG)Y
48

V
96
G (VPGVG)Y
96

V
144
G (VPGVG)Y
144

V
192
G (VPGVG)Y
192

x

ABSTRACT
Dry eye syndrome is a common disorder of the tear film characterized by decreased
tear production, normally treated using conventional drops containing small molecule
drugs which wash away from the eye within minutes. Due to rapid clearance, ocular drug
formulations must be given frequently. The main aim of this project is to design a
sustained release protein which will be retained in the eye for a longer period of time. In
order to achieve this, we have constructed a fusion protein between Elastin-like
polypeptides (ELPs) which has a unique characteristic inverse phase transition
temperature (T
t
) and lacritin which is a protein proposed to stimulate tear secretion.
The ELP library with valine as the guest residue has been characterized using the
UV-vis spectrophotometer. The 96 pentameric ELP with valine as the guest residue at
various concentrations shows a range of T
t
which is between room temperature and body
temperature. This is most favorable for instillation into the eye and has been used in the
formation of the fusion protein. The effect of rabbit tear enzymes on the ELPs has been
evaluated and it is observed that over a time interval of 2 hours there is no significant
degradation of the ELP, which makes it appropriate for ocular application.
1

CHAPTER 1

INTRODUCTION
Dry eye syndrome (DES) is a disorder which causes excessive tear evaporation
leading to several problems of the eye such as ocular discomfort, inflammation, itching
and a reduction in visual acuity [18, 27, 31]. This is a common disorder which is more
prevalent among elderly and postmenopausal women than men [7, 36]. There are several
formulations available in the market to treat DES by hydrating the tear film. These ocular
drug formulations need to be given frequently, every 2 to 8 hours as they readily wash
away from the eyes.
Elastin-like polypeptides are a group of biopolymers which have a specific transition
temperature above which they form an aggregate [4, 8]. Our hypothesis is that these
polymers will be retained in the eye for a long period of time provided that the transition
temperature is adjusted below the body temperature. An ELP formulation capable of
delivering a drug to the eye for an extended period may provide a mechanism for
overcoming ocular dry eye disorders.
2

To treat this syndrome more effectively, we have designed a fusion protein
between the ELP and lacritin, a secreted glycoprotein present in tears recently found to
stimulate the production of tears in rabbits [34].
This fusion protein may be more effective than conventional eye drops since they
have tear stimulating properties of lacritin and can be retained in the eye for a longer
period of time as compared to conventional drops due to the aggregation of the ELPs.

3

CHAPTER 2

BACKGROUND
2.1 The tear film
Mucous, aqueous, and lipid layers make up the tear film each of which have
specific roles in maintaining ocular health [30]. The mucous layer is the innermost part of
the tear film, responsible for lubricating the ocular surface and getting rid of foreign
matter like microbes, debris etc [14]. The lacrimal glands are responsible for the
production of the aqueous layer [30] which consists of water, electrolytes, IgA, lyzosyme,
lactoferrin [26]. It is responsible for maintaining the ocular surface by hydrating it,
providing oxygen and promoting wound healing [14, 17]. The outermost layer consists of
the lipid film which reduces tear evaporation and increases the spreading of the tear film
[2, 5, 12, 14].
These three layers are thus responsible for keeping the ocular surface healthy by
lubricating it, getting rid of foreign matter, providing nutrients and antibacterial
4

substances to protect the ocular surface [26]. Ocular problems could arise from
deficiency of any of the three layers.
One of the most common disorders caused by excessive evaporation of the tear
film is known as Dry eye syndrome [18, 36].

2.2 Dry eye syndrome
Dry eye syndrome (DES) also known as keratoconjunctivitis sicca or
dysfunctional tear syndrome [3]

is a disorder characterized by several symptoms such as
increased osmolarity of tear film, visual problems and discomfort, burning, itching and
inflammation of the eye [24, 27, 31]. However, the patients symptoms are not always the
same as the clinical observations [33]. A number of eye drops in the market are available
which would only hydrate the tear film to treat this syndrome. This does not treat dry
eyes completely as there are several different factors contributing to this disorder.
There are two major reasons for DES which could be aqueous deficient or
evaporative [24]. Sjögren's syndrome is one of the causes of aqueous tear-deficient dry
eyes, resulting in dry eye or dry mouth due to its affect on lacrimal and salivary glands
5

[11, 44]. Other causes for aqueous deficient dry eyes are age related, due to lacrimal
deficiency, menopause, and certain systemic drugs [24].

The various causes for
evaporative DES are eyelid and blinking disorders, meibomian gland disease [18, 24].
Changes in the epithelial structure of the cornea and conjunctiva, reduction in tear
secretion and stability, alterations in blinking activity are few of the other reasons for
DES [28]. Tear film osmolarity is also an important determinant of DES. An increase in
the osmolarity of the tear fluid could be an indication of a decrease in tear production
since a decrease in the rate of lacrimal gland secretion causes an increase in the
osmolarity of the lacrimal gland fluid [15].
For a more effective treatment, a combination of an increase in tear secretion,
hydration of tear film and sustained release of the drug would be more beneficial than
just lubrication of the eye.

6

2.3 Elastin–like polypeptides
There have been several advances in the use of protein based polymers for drug
targeting [8].

The properties of these protein based polymers can be manipulated to
confer essential characteristics for targeting [40].

These proteins can be degraded by the
normal protein turnover pathway or utilized by normal physiological processes of the
body [8]. They can be used as drug carriers and have the advantages of controlled release,
site-directed delivery and site avoidance delivery.

There can be significantly lower
systemic drug toxicity as compared to the free drug due to the use of these polymer
systems [32].
Elastin-like polypeptides (ELPs) are a type of thermally responsive protein based
polymers composed of a pentapeptide repeat (VPGXG) where X is any guest residue
which is responsible for the phase behavior properties of the ELPs [8]. They belong to a
group of smart polypeptide polymers inspired from human elastin [43]. These
biopolymers have a unique characteristic inverse phase transition temperature, such that
they are soluble in aqueous solutions below their transition temperature but collapse and
7

aggregate under hydrophobic forces at temperatures above their critical transition
temperature [4, 8].

(Fig.1)

Fig.1 Phase transition of ELPs: ELPs form light-scattering aggregates (right) when they are
heated above their transition temperature and can be reversibly converted into soluble phase (left)
on cooling

The transition temperature (T
t
) of the ELP can be modified by changing the amino
acid at the Xaa position or the molecular weight. An increase in the hydrophobicity of the
guest residue (Xaa) can reduce the T
t
of the ELP [39, 41]. The amino acid sequence and
the molecular weight of the ELPs are important for the pharmacokinetics, biological
8

activity and biodegradation of the polypeptide [13, 25]. The chain length and ELP
concentration are found to be inversely proportional to the T
t
of the ELP [8]. This
property can be utilized to manipulate the T
t
of the ELPs. ELPs also have unique
properties that promote phase separation, recombinant expression, protein purification,
and self-assembly of nanostructures [10, 43].
ELPs can incorporate drugs by three different methods, by conjugation, chelation
or encapsulation [8].

Proteolytic linkages can also be incorporated between the polymer
and the drug which can be cleaved by appropriate enzymes [8]. Depending on their use,
these ELP drug carriers can be divided into three main types: ELP homopolymers for the
delivery of chemically attached drugs, block copolymers that can form micelles for the
encapsulation of drugs and fusion proteins [8].

2.3.1 Recombinant Synthesis of the ELP library [8]
ELPs are synthesized using a technique known as Recursive Directional Ligation
(RDL). The synthesis of the ELPs by this recombinant genetic technique provides a good
9

control over the ELP sequence and molecular weight [32]. The RDL technique produces
repetitive polypeptides of specific chain lengths.

Fig.2 Steps involved in RDL: The ELP with n chain length is inserted into a cloning vector. It is
further digested using both restriction enzymes (A and B) to produce a monomer insert as well as
with only restriction enzyme A to produce a linearized plasmid which is ligated with the insert.
This produces a dimerized ELP gene with a 2n chain length (2a). These ligations and digestions
are repeated i number of times to give increasing chain lengths of the ELPs (2b)

An ELP gene is inserted into a modified cloning vector such as pET25b+ which
has an additional BseRI cut site. The monomer gene is obtained by digestion at sites A
and B with appropriate restriction enzymes. The linearized plasmid containing ELP is
10

obtained by digesting at site A. (Fig.2a) The ELP insert is ligated with the plasmid
containing the ELP leading to dimerization of the gene. These Ligation/digestion cycles
are repeated until the desired ELP chain length is obtained. (Fig 2b)

2.3.2 Purification of ELPs using Inverse transition cycling [9, 22]

After the synthesis, this library of ELPs is purified from bacterial cell lysate using
Inverse phase transition for Protein purification.

Fig.3 Inverse transition cycling (ITC): ELPs form an aggregate on raising the temperature or by
adding different salts. These aggregates can be separated by centrifugation. The supernatant is
discarded to get rid of bacterial impurities. The aggregate is re suspended in PBS and solution
temperature is dropped. On repeating these cycles several times, purified ELPs are obtained

11

ITC is used to purify ELPs that have been expressed and released from bacteria.
ELPs are purified out of bacterial cell lysates by raising the solution temperature and/or
by adding salt such as NaCl/ Ammonium sulfate. This results in the formation of ELP
aggregates that can be collected by centrifugation. The supernatant is then discarded,
which removes soluble contaminant macromolecules. The remaining ELP pellet is
resolubilized in PBS buffer at a temperature below T
t
. The process is repeated until the
desired purity is achieved. The purified proteins are further characterized using the
DU800 UV-vis spectrophotometer and SDS-PAGE to confirm the purity of the protein.
This technique is relatively economical and does not require the use of complex
instruments or special reagents.

2.4 Lacritin
The lacrimal functional unit (LFU) is composed of the lacrimal glands, cornea,
meibomian glands, motor and sensory neurons along with eye lids [37]. There are several
LFU proteins in different tissues. Lacritin is one of the proteins of the LFU which is
12

highly expressed in the lacrimal glands, detected in lacrimal acinar cell secretory granules
[21, 35]

and also found to be down regulated in dry eye syndromes [6].

2.4.1 Structural attributes of lacritin
Lacritin is a 12.3 kDa secreted glycoprotein in tears with a 417bp open reading
frame [35]. It shows an N-glycosylation site near the C-terminus and 6 O-glycosylation
sites [35]. Lacritin targets only epithelia and not the other cell types [42]. It is a
stimulated secretory product of acinar cells and implies an autocrine or paracrine
mechanism possibly due to the luminal acinar cell receptors [35]. It has an effect only on
unstimulated secretion of the acinar cells [35].
Lacritin is found to have an abnormally slow migration of around 18 kDa as
opposed to its original size of 12.3 kDa [21]. This was analyzed in various studies to be
due to the disordered and ordered N and C-terminals of lacritin respectively [21]. Splice
variants of lacritin-b, lacritin-c [20] and lacritin-d [29] have been documented of which
lacritin-a is found to be the most common one. These forms have differences in their
glycosylation site and also differ slightly in their sequence [21].

Lacritin-b and c due to
13

their differences in sequence are assumed to be inactive where as not much about lacritin-
d is predicted [21].
A targeting mechanism of lacritin has been previously studied using recombinant
proteins [42]. The C-terminal alpha-helix part of lacritin targets a common heparan
sulfate proteoglycan that surrounds the cornea known as Syndecan-1(SDC-1) [16], the
binding of which requires the 3 heparan sulfate bonds to be cleaved off [19] (Fig.4).
Heparanase is found in tears, which causes the cleavage and exposes the lacritin binding
region of SDC-1 [19] which is found to be restricted to the initial 51 N-terminus amino
acids [21]. This binding sets out a cascade of events which are essential for its mitogenic
activity, cell survival along with protein translation [21].

14

Fig.4 Binding of Lacritin onto syndecan-1: Cleavage of the 3 heparan sulfate bonds of SDC-1
with heparanase exposes binding region of lacritin which is restricted only to the first 51 amino
acids of syndecan-1

2.4.2 Implication in various diseases
Lacritin is found to increase basal tear secretion when topically applied in rabbits
[34]. Although the prosecretory mechanism of lacritin on the eye is yet to be known,
lacritin in corneal epithelia cell culture is found to increase MUC16 which is an ocular
surface mucin responsible for lubricating and protecting the eye. This could be one of the
topical uses of lacritin [21]. Lacritin may also have a beneficial role in patients suffering
from blepharitis which is caused due to an increased viscosity of lipid secretion by
15

meibomian glands leading to bacterial infection [6]. It has protective action against
inflammatory cytokines, interferon γ and TNF and hence beneficial against inflammation
of the eye [21].

2.5 Synthesis of lacritin-ELP fusion protein
ELPs are synthesized as mentioned above by a technique known as RDL to
produce the desired chain length. After this step, the plasmids containing lacritin and ELP
are digested using the same restriction enzymes. The two fragments are ligated together
in such a manner that lacritin gene is at the N-terminus (Fig. 5). This plasmid containing
the fusion gene will be further purified and transformed into an expression host such as
BLR cells which will express the fusion protein. This fusion protein is purified from the
bacterial cell lysates by Inverse transition cycling.
16

Fig.5 Synthesis of Lacritin-ELP fusion protein: BseRI and BssHII enzymes are selected to
digest lacritin and ELP fragments from their respective vectors which are ligated to give the
fusion gene.This is further transformed into BLR cells for the expression of the fusion protein

2.6 Specific Aims
Lacritin is proposed to be useful for the treatment of dry eye syndrome by its
ability to promote tear secretion [35].

Lacritin in the form of conventional eye drops
would wash away from the eye within minutes. The specific aim of this project is to
develop an innovative strategy to retain the drug in the eye for a longer period of time.
This can be achieved by developing a sustained release formulation. For this purpose, an
appropriate ELP is chosen which would transition between room temperature and body
17

temperature. The 96 pentapeptide ELP with valine (V
96
) as the guest residue has a
transition temperature of around 32.3 °C at 25 μM concentration as shown in Fig. 6.
This ELP has been selected for the synthesis of a fusion protein. Lacritin fused to
the V
96
is assumed to form a sustained release product which will have slow clearance in
the eye and be retained for a longer period of time. This will result in higher patient
compliance, as the frequency of administration of the drops will be reduced. A thrombin
cleavage site has been incorporated into the lacritin sequence to obtain free lacritin for
comparative studies.

Fig.6 Turbidity plot for ELP, G (VPGVG)
96
Y: From the plot it is evident that as the
concentration increases from 5 μM to 100 μM the transition temperature decreases
18

CHAPTER 3

MATERIALS AND METHODS
3.1 Materials
The lacritin minigene was obtained from Integrated DNA Technology in the
pIDTSMART-KAN vector. The ELP library with valine as the guest residue was
synthesized by Pang-Yu Hsueh from Dr. Sarah Hamm-Alvarez laboratory. All the
enzymes and buffers were obtained from New England Biolabs
®
Inc. unless otherwise
stated.

3.2 Design of Lacritin-ELP using Laser gene
The sequence of lacritin was obtained from published literature [35] . (Lacritin
sequence is shown in red in Table 1). DNASTAR Lasergene was used to design the
sequence of the lacritin-ELP fusion protein in the pET25b+ vector. The lacritin sequence
was modified to incorporate a thrombin cleavage site (underlined in Table 1) in order to
release lacritin from the ELP. The fusion protein was designed to incorporate the 96
19

pentapeptide ELP with valine as the guest residue. The calculated molecular weight was
52.5 kDa and predicted T
t
was 30 °C from the formula T
t
=T
t, c
+k/L ln (C
c
/C) at 25 μM of
the ELP [23, 40]. This is expected to give slow clearance as summarized in Table 1.

Table 1 Construct of ELP and Lacritin-ELP: Lacritin-ELP sequence containing the 96
pentameric valine ELP has a predicted M.W. of 52.5 kDa and slow ocular clearance. Y is
incorporated at the end of the sequence for quantification using a UV-vis spectrophotometer

Guest
residue
(X)
ELP
amino
acid
Sequence
MW
(kDa)
Lacritin-ELP amino acid
sequence
MW
(kDa)
T
t

(°C)
Expected
ocular
clearance
V
MG[VPG
VG]
96
Y
39.5
HMGEDASSDSTGADPA
QEAGTSKPNEEISGPAEP
ASPPETTTTAQETSAAA
VQGTAKVTSSRQELNPL
KSIVEKSILLTEQALAKA
GKGMHGGVPGGKQFIE
NGSEFAQKLLKKFSLLK
PWA-GLVPR|GS-
G[VPGVG]
96
Y
52.5 30 slow

20

Fig.7 Lacritin sequence: Lacritin consists of 119 amino acids, 6 O-glycosylation sites and 3 N
glycosylation sites along with an incorporated thrombin cleavage site. Several restrictions
enzymes are present in this sequence such as NdeI, BamHI and BseRI. These enzymes are used
for diagnostic digests and also for the insertion of the ELP gene

The complete lacritin sequence is shown in Fig.7 from Glutamic acid (E) to
Alanine (A) (shown in a block). Lacritin consists of 119 amino acids without the signal
peptide. There is a thrombin recognition sequence and a BseRI cut site where the V
96

ELP is ligated. The blocks in green signify O-glycosylation and the blocks in red signify
21

N-glycosylation, which are not synthesized using bacterial expression, nor are they
required for activity [35, 42].

3.3 Recombinant synthesis of Lacritin into pET25b+ vector
Lacritin minigene and pET25b+ (Novagen
®
) were transformed into E. coli Top 10
cells (Invitrogen™) and plated onto kanamycin and ampicillin resistant plates
respectively. These plates were kept in an incubator overnight at 37 °C. Colonies were
selected from each of the plates and inoculated into TB culture media along with 4 μl of
100 mg/ml kanamycin and amipicillin respectively. These culture tubes were place
overnight in the shaker at 37 °C. The overnight culture was further purified (QIAprep
Spin Miniprep Kit, QIAGEN).
Digestion: Two unique restriction sites were selected in order to digest lacritin from the
pIDTSMART-KAN vector to put it into the pET25b+. Lacritin as well as pET25b+ were
digested using 1 μl of BamHI and NdeI in the presence of buffer NEB4 and 10X BSA for
3 hours at 37 °C. After digestion, pET25b+ vector was treated with alkaline phosphatase
(CIP) for 1 hour at 37 °C to prevent the sticky ends of the vector from ligating.
22

Agarose gel electrophoresis: Digested lacritin was loaded onto 1% low melt Agarose gel
(Bio-rad Cat# 161-3100) along with bromophenol blue loading dye. The gel was
analyzed using the Typhoon 8610(variable mode imager). The lacritin band of around
402 base pairs was selected and gel purified (QIAquick Gel Extraction Kit, QIAGEN).
CIPed vector was also spin column purified (QIAquick Gel Extraction Kit, QIAGEN).
Ligation: Gel purified lacritin and pET25b+ were ligated with a 2:1 insert to vector ratio
respectively using T4 DNA ligase(Invitrogen™) and ligase buffer. This was further
transformed into E.coli TOP10 cells and plated onto ampicillin plates. Colonies were
selected the next day which were inoculated and purified. The sequence of lacritin into
pET25b+ was confirmed from the DNA sequencing results provided by the Norris DNA
core facility, USC. The T7 promoter and T7 terminator primers were used for obtaining
the sequencing results.

3.4 Recombinant synthesis of V
96
into the pET25b+ vector containing Lacritin
DMSO stock of the V
96
DNA in the pET25b+ was inoculated and purified.
(QIAprep Spin Miniprep Kit, QIAGEN)
23

Digestion: Lacritin in pET25b+ along with the V
96
in pET25b+ were digested using two
unique site enzymes which were BseRI and BssHII for 3 hours at 37 °C.
Agarose Gel electrophoresis: The digested DNA was loaded on agarose gel and the
appropriate bands containing lacritin (1620 bps) and V
96
(5676 bp) were selected which
were further gel purified.
Ligation: Lacritin and V
96
were ligated using T4 DNA ligase and ligase buffer
(Invitrogen™) with a ratio of 1:1 respectively. This was further transformed into TOP10
cells and plated onto ampicillin plates. Colonies were selected the next day which were
inoculated and purified. The sequence of lacritin fused to V
96
was confirmed by the
Norris DNA core facility, USC.

3.5 Protein purification using ITC [9]
The purified plasmid containing the Lacritin-V
96
sequence was transformed into
BLR cells. A colony was selected and cultured in TB media. The overnight culture was
centrifuged at 3000 rpm, 4 C for 10 minutes. The supernatant was discarded and the
pellet was resolubilized in filtered PBS. The re suspended pellet was inoculated in culture
24

media and kept in the incubator overnight at 37 °C , 250 rpm. After overnight incubation,
the culture was centrifuged at 3000 rpm, 4 °C for 10 minutes (Sorvall
®
RC 3C Plus). The
supernatant containing the ELP was resuspended in cold PBS. After this the suspension
was sonicated to lyse the cells.
Cold Spin: The sonicated product was subjected to a cold spin at 12000 rpm, 4 °C for 15
minutes. The supernatant contained the ELP whereas the pellet contained the insoluble
debris. Poly ethyleneimine (Aldrich Chemistry Cat#408700-1L) was added to a final
concentration of 0.5% and the solution was incubated for 10-20 minutes on ice. The
solution was again centrifuged at 12000 rpm for 15 minutes at 4 °C to get rid of the
precipitated DNA and insoluble cellular debris in the pellet. The supernatant was placed
in a 37 °C water bath for 10 minutes along with the addition of 5 gm of ammonium
sulfate to reduce the T
t
of the ELP.
Hot Spin: The solution was then centrifuged at 37 °C for 10 minutes, 4000 rpm. The
supernatant was discarded and the pellet containing the ELP was re-suspended on ice in
15 ml of PBS.

25

Cold Spin: The suspension was again centrifuged at 12000 rpm at 4 °C for 10 minutes to
remove remaining debris and insoluble matter. The Hot and Cold Spins were repeated at
least three times until a high level of purity of the pellet containing the ELP was
achieved.

3.6 Beckman Coulter DU 800 UV-vis spectrophotometer [23]
The ELP Valine library (V
24
,V
36
,V
48
,V
96
,V
144
,V
192
) transition temperatures were
characterized over a range of concentrations (5, 10, 25, 50, and 100 μM ELP in phosphate
buffer) by raising the temperature at 1 °C/min on a Beckman DU800 UV-vis
spectrophotometer equipped with a multicell Peltier temperature controller. The T
t
of V
96
over the same range of concentrations was also evaluated in the presence of rabbit tears
and PBS. The transition temperature is defined at the maximum first derivative of the
optical density at 350 nm. This data was used to compare the T
t
of the different lengths of
ELPs with respect to their concentration.

26

3.7 SDS-PAGE
PAGEr
®
GOLD Precast 4-20% gradient gels (LONZA) were used to check the
purity of the ELPs. 4X sodium dodecyl sulfate (SDS) loading buffer consisting of
glycerol and bromophenol blue was added to the ELPs after which the proteins were
denatured at 95 °C for 5 minutes. 40 μg of the ELPs were loaded into the wells. The
ELPs were stained with copper chloride as they do not stain with commassie blue [38].
Kaleidoscope protein ladder (Bio-rad laboratories) was used as the protein molecular
weight marker. The Chemi-doc instrument (Bio-rad laboratories) was used to analyze
these gels.

3.8 Degradation studies
G (VPGVG)
96
Y, was incubated for time intervals (0, 5,15, 30, 60 minutes) in
rabbit tears at 37 ºC. The Experion (Bio-rad laboratories) along with the protein analysis
kit was used to obtain the protein results.

27

3.9 Thrombin cleavage assay
The lacritin-V
96
fusion protein consists of a thrombin cleavage site
(LeuValProArg|GlySer) in order to separate the lacritin from the fusion protein for
comparative studies. Thrombin kit (Novagen
®
) was used to perform the cleavage assay
for the fusion protein. 30 µg of the fusion protein was incubated with 0.5 units of
thrombin along with 15 µl of 1x cleavage buffer. The mixture was incubated for time
intervals (20, 60, 120, 240 minutes) at 37 ºC. The mixture was subsequently loaded onto
SDS-PAGE for the detection of thrombin cleavage of the fusion protein.

28

CHAPTER 4

RESULTS
4.1 Characterization of the Valine Library using UV-vis spectrophotometer
Different chain lengths (Fig.6, Fig.9 - Fig.13) of the Valine library of ELPs were
characterized at 5 fixed concentrations (5, 10, 25, 50, 100 μM in PBS buffer). It is
observed that as the chain length increases from V
24
to V
192
, the transition temperature of
the ELPs decreases (Fig.8). Also as the concentration increases from 5 μM to 100 μM the
transition temperature decreases. (Fig.8) This observation is consistent for all the chain
lengths of the ELPs except for V
24
which shows a lower T
t
of 67.6 °C at 5 μM as
compared to 78 °C at 10 μM (Table 3). From Fig. 9 it is apparent that at 5 μM and 10
μM, V
24
does not show proper peaks implying that it has not transitioned.
Fig.8 is a summary of the different ELP chain lengths showing that the transition
temperature is inversely proportional to the chain length as well as the concentration.
These results confirm the fact that by manipulating the chain length, molecular weight as
well as the concentration, ELPs of desired transition temperature can be conveniently
29

synthesized. V
96
has been selected for all the experiments as it has its transition
temperature above room temperature and can easily form an aggregate upon instillation
into the eye, since the temperature of the eye is around 37 °C.

Fig.8 Log plot of the turbidity of different ELP chain lengths at various
concentrations: From the graph it is seen that as the chain length and concentration increases
from V
24
-V
192
the transition temperature decreases.V
24
does not provide clear data for its T
t

30

Fig.9 Turbidity plot for ELP, G (VPGVG)
24
Y: From the plot it is evident that as the
concentration increases the transition temperature decreases. However at 5, 10 μM there is no
sharp peak and the transition temperature cannot be accurately interpreted

Fig.10 Turbidity plot for ELP, G (VPGVG)
36
Y: From the plot it is evident that as the
concentration increases the transition temperature decreases.
31

Fig.11 Turbidity plot for ELP, G (VPGVG)
48
Y: From the plot it is evident that as the
concentration increases the transition temperature decreases

Fig.12 Turbidity plot for ELP, G (VPGVG)
144
Y: From the plot it is evident that as the
concentration increases the transition temperature decreases
32

Fig.13 Turbidity plot for ELP, G (VPGVG)
192
Y: From the plot it is evident that as the
concentration increases the transition temperature decreases

A comparison has been made between the predicated and experimental T
t
in Table
2 and Table 3 using the formula T
t
=T
t, c
+k/L ln (C
c
/C) for the predicted T
t
where T
t, c
is
the critical T
t
, C
c
is the critical concentration, C is the ELP concentration, k is a constant,
L is the chain length of the ELP

[23, 40]. This comparison is important in determining
the accuracy of the experimental T
t
when compared to the predicted values. It is observed
that for most of the concentrations and chain lengths of the valine ELP library, the
predicted and calculated T
t
are almost similar. However V
24
at 25 μM and 10 μM show
considerable differences between the predicated and calculated T
t
. The above formula can
33

be used to predict the T
t
for any chain length of the ELP with valine as the guest residue
without performing any experiments making it easier to select the appropriate chain
length for a specific T
t
.

Table 2 Summary of the predicted T
t
for the valine library:
a
T
t
is the predicted T
t
using the
formula T
t
=T
t, c
+k/L ln (C
c
/C)

[23]

Concentration
M
a
T
t
of V
24
( C)
a
T
t
of V
36
( C)
a
T
t
of V
48
( C)
a
T
t
of V
96
( C)
a
T
t
of V
144
( C)
a
T
t
of V
192
( C)
100 51.6 41.4 36.2 28.5 25.9 24.7
50 55.4 43.9 38.1 29.4 26.6 25.1
25 58.7 46 39.7 30 27.11 25.5
10 64.1 49.7 42.5 31.6 28 26.2
5 67.9 52.2 44.4 32.6 28.7 26.7
34

Table 3 Summary of the experimental T
t
for the valine library:
b
T
t
is the experimental T
t
using
the Beckman Coulter DU 800 UV-vis spectrophotometer [23]

Concentration
M
b
T
t
of V
24
( C)
b
T
t
of V
36
( C)
b
T
t
of V
48
( C)
b
T
t
of V
96
( C)
b
T
t
of V
144
( C)
b
T
t
of V
192
( C)
100 57.9 44.2 36.7 30.6 28.1 26.8
50 63.2 46 38.4 31.6 29.3 27.8
25 71.2 48.6 38.9 32.3 30.5 28.6
10 78 52.4 41.9 32.8 31.7 29.8
5 67.6 59.2 45.7 33.1 32.6 30
35

Table 4 ELP constructs along with their predicted ocular clearance: All the ELPs with 96
pentapeptides or longer chain lengths are predicted to have slow clearance and can be retained in
the eyes for a longer period of time

ELP amino acid
Sequence
MW(kDa) T
t
for 25 µM
(°C)
Expected
ocular
clearance
MG[VPGVG]
24
Y 10 71.2 Fast
MG[VPGVG]
36
Y 14.9 48.6 Fast
MG[VPGVG]
48
Y 19.9 38.9 Fast
MG[VPGVG]
96
Y 39.5 32.3 slow
MG[VPGVG]
144
Y 59.2 30.5 slow

4.2 Confirmation of the purity and molecular weight of the ELPs
Fig.14 is an SDS-PAGE of the valine library (V
24
, V
36
, V
48
, V
96
, V
144
, V
192
)
confirming their respective molecular weights to be around 10, 14.9, 19.9, 39.5, 59.2,
78.8 kDa respectively. 40 μg of the ELPs were loaded into the gel. The bands appear to
be thick and clear for all the libraries except for V
144
which shows two bands at around
50 kDa and slightly above 50 kDa suggesting the presence of cellular debris and
impurities. Further rounds of ITC would help in eliminating the debris and increasing the
purity of the protein.
36

Fig.14 SDS-PAGE of Valine library proteins: The molecular weight of all the ELP chain lengths
seems accurate except for V
144
which shows two bands suggesting the presence of impurities

4.3 Degradation of ELPs in physiological fluids
A 39.5 kDa ELP, G (VPGVG)
96
Y, was incubated for time intervals (0, 5, 15, 30,
60 minutes) in rabbit tears at 37 ºC. A light band is observed at around 37 kDa
throughout the time interval which appears to be the ELP. There are bands above the
ELP, which are tear proteins as can be confirmed by looking at the lane with only tears in
Fig.15. Few bands appear to be below the ELP. ELP bands are consistent throughout the
37

time interval; it is assumed that no degradation products are observed over this interval.
However, concentrated tear proteins could partially obscure this assay and it is important
to perform further studies to confirm this result.

Fig.15 SDS Page of ELPs incubated in tears: A consistent clear band is observed at 37 kDa
which is the ELP. No degradation products are observed for the ELP over time length of 60
minutes

4.4 Comparison of T
t
for V
96
in PBS and tears
The T
t
for a 39.5 kDa ELP, G (VPGVG)
96
Y , at various concentrations (5, 10, 25,
50, 100 μM) was evaluated in phosphate buffered saline (PBS) and rabbit tears. Fig.16 is
38

a log plot of the concentration. The T
t
in PBS and tears are similar which suggests that
there is no effect of the tear proteins and enzymes on T
t
of the ELP.

Fig.16 Concentration dependence of T
t
in physiological solutions: The T
t
of the ELP is similar
in tears and saline

4.5 Confirmation of lacritin and fusion protein sequence in pET25b+
The presence of lacritin in the pET25b+ vector was confirmed by digesting the
vector with unique site endonucleases: XbaI and Xho1 along with NEB4 and 10X BSA
39

buffers for 3 hours at 37 °C. The expected base pair is around 482 bps. The bands are
observed at around 500 bp which suggests the presence of lacritin in the vector (Fig.17).

Fig.17 Agarose Gel for diagnostic digest of lacritin: Both the lacritin samples show the expected
bands (500bps) suggesting the presence of lacritin in pET25b+ vector

The lacritin-V
96
fusion gene was also digested for 3 hours at 37 °C with unique
site endonucleases: XbaI and Xho1 along with NEB4 and 10X BSA buffers. Fig.18
shows an agarose gel of the digested plasmid. There appear to be two bands. The
sequence of the entire plasmid is around 7301 bps and the sequence from XbaI to XhoI is
40

around 1922 bps which corresponds to the second band. This result confirms that the
plasmid contains both lacritin and the ELP.

Fig.18 Agarose Gel for diagnostic digest of Lacritin-V
96
: The digested second band of Lac-V
96

appears to be at the correct base pair suggesting the presence of lacritin -V
96
in the pET25b+
vector

4.6 Characterization of Lacritin-V
96
using UV-vis spectrophotometer
The recombinant fusion protein of lacritin and V
96
was characterized at 5 fixed
concentrations (5, 10, 25, 50, 100 μM in PBS buffer) using the DU 800 UV-vis
spectrophotometer. The T
t
of the fusion protein at 5, 10 μM concentrations is similar to
41

that of V
96
alone (Table 3, 5). However at 25, 50,100 μM the T
t
appears to have reduced
compared to the T
t
of V
96
(Table 3, 5).

Fig.19 Turbidity plot of Lacritin-V
96
: From the plot it is evident that as the concentration
increases the transition temperature decreases. The T
t
at 10 μM is higher than that at 5 μM
Table 5 Summary of the T
t
for the fusion protein: As the Concentration decreases the T
t
increases
Concentration (μM) Temperature (°C)
100 30.0
50 30.4
25 30.7
10 39.6
5 35.6

42

4.7 SDS-Page of fusion protein
Lacritin has a molecular weight of around 12.3 kDa. With the addition of the
thrombin cleavage sequence, the molecular weight is around 13 kDa. Studies have shown
that lacritin has anomalous migration in SDS-PAGE and runs above its molecular weight
due to its disordered domain [21]. The molecular weight of the V
96
is around 39.5 kDa
(Table 1). The total M.W. of the fusion protein is expected to be around 52.5 kDa. Fig.20
shows bands above 50 kDa which should be the fusion protein. There is a slight band
around 20 kDa which could be the cleaved off lacritin. It is assumed that during rounds of
ITC some of the lacritin protein is getting cleaved off. These results are confirmed further
in Fig. 21.
43

Fig.20 SDS-PAGE of the fusion protein with coomassie staining: At both the concentrations
(25,50 μM) of the fusion protein, the bands appear to be at around 52 kDa which is the correct
size of the protein. There are light bands at around 18 kDa which could be the cleaved off lacritin

ELPs stain only with copper chloride [38]. Fig.21 shows two bands for the fusion
protein one at around 52 kDa and the other at around 39 kDa which could be the ELP.
This confirms that some of the fusion protein is cleaving off to form lacritin and ELP
which is either due to excessive salt addition or rise in temperature during the ITC
purification.

44

Fig.21 SDS-PAGE of the fusion protein with copper chloride staining: The upper band of the
100 µM sample is the fusion protein, whereas the lower band is the cleaved off ELP which could
be due to separation of lacritin from ELP during the hot spin of the ITC

4.8 Thrombin cleavage assay
The fusion protein was incubated with thrombin to cleave off the lacritin from the
fusion protein. In Fig.22 there is a band observed at around 52 kDa which is the fusion
protein. There are two other bands observed at around 25 kDa and another at around 18
kDa. The band at 18 kDa could be lacritin. Although lacritin has a M.W. of around 13
kDa it is observed to show anomalous migration of 18 kDa in SDS-PAGE [21, 35]. The
second band at around 25 kDa could be an impurity or another fragment of the fusion
45

protein. The band at 37 kDa is thrombin. As the time increases from 30 minutes to 240
minutes, the band at 52 kDa appear to become lighter suggesting the cleavage of lacritin-
V
96
fusion protein in the presence of thrombin.

Fig.22 Cleavage of fusion protein using thrombin: The fusion protein was incubated with
thrombin over a time interval of 2 hours. From the figure it is observed that lacritin is getting
cleaved off. There is an unknown degradation product observed at 25 kDa

46

CHAPTER 5

DISCUSSION AND CONCLUSION
Different chain lengths of Elastin like polypeptides can be synthesized using
recombinant genetic techniques such as recursive direction ligation [8]. A 96
pentapeptide ELP with valine as the guest residue has a T
t
of around 32.3 C at 25 μM
(Table 3). Since this falls into the range between room and body temperature, the V
96

ELP can be expected to form an aggregate as soon as it is instilled into the eye. The T
t
of
this ELP was tested in different physiological fluids such as tears and PBS (Fig.16). It is
observed that there is no significant change in the T
t
of the ELP in these fluids. ELPs
were also digested over a time interval of 2 hours in the presence of tears to test the effect
of various enzymes in the physiological fluids on the ELP protein. From the results
obtained there is no significant cleavage of the ELP (Fig. 15). Both the results suggest
that ELPs will be stable in tears.
Lacritin is a recently discovered protein of the LFU which is found to promote
tear secretion and is useful in the treatment of several eye disorders [35]. The aim of this
47

project is to develop a fusion protein between the lacritin and ELP in order to retain it in
the eye for a longer period of time. ELPs are biodegradable and inert which would not
affect the ocular surface [8].
An SDS-PAGE of the fusion protein confirmed its molecular weight to be at
around 52.5 kDa (Fig.21) although there seem to be a few degradation products which are
assuming to be the cleaved off lacritin and ELP. This could be due to degradation during
the purification process. The T
t
of the fusion protein at 25 M is 30.7 °C and T
t
of V
96
alone is 32.3 °C which are almost similar, implying that the addition of lacritin does not
significantly change the T
t
of the ELP. The T
t
at various concentrations appears to be
within the desired range for instillation into the eye.
Further studies were done on the fusion protein in the presence of thrombin to
confirm the thrombin cleavage sequence in the fusion protein. The results from the SDS-
PAGE (Fig.22) show that there is a band at around 52.5 kDa which is the fusion protein.
There appear to be two more bands at around 25 kDa and 18 kDa. The band at 18 kDa is
thought to be lacritin, which has anomalous migration in SDS-PAGE [35]. The band at
25 kDa could be an additional cleavage product or an impurity.
48

CHAPTER 6

FUTURE DIRECTIONS
Purification method of the fusion protein needs to be modified to prevent the
cleavage of lacritin from the ELP. This can be achieved by lowering the temperature
during the Hot Spin since changes in temperature can lead to denaturation of the protein
causing lacritin to be released from the ELP [9]. Heat may promote proteolysis via trace
enzymes.
Further cell function assays need to be performed to test the secretory activity of
the fusion protein on the rabbit lacrimal acinar cells. The β-hexosaminidase assay can be
performed in the presence of the fusion protein to determine its secretory activity. A
comparison between free lacritin and the fusion protein needs to be made to test its
efficacy in the treatment of dry eye syndrome.
Although lacritin is found to promote rat lacrimal acinar cell secretion, its
mechanism in promoting topical tear secretion needs to be well elucidated to confirm its
role in treatment of dry eye syndrome [21].The complexity of lacritin in promoting
49

topical tear secretion can to be studied further by understanding its ocular cell signaling
and targeting mechanisms.
In vivo assays on rabbit tears should also be performed to check the increased
secretion of tears using the Schirmer tear test [1].

50

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Abstract (if available)

Abstract Dry eye syndrome is a common disorder of the tear film characterized by decreased tear production, normally treated using conventional drops containing small molecule drugs which wash away from the eye within minutes. Due to rapid clearance, ocular drug formulations must be given frequently. The main aim of this project is to design a sustained release protein which will be retained in the eye for a longer period of time. In order to achieve this, we have constructed a fusion protein between Elastin-like polypeptides (ELPs) which has a unique characteristic inverse phase transition temperature (Tt) and lacritin which is a protein proposed to stimulate tear secretion.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Jashnani, Aarti (author)

Core Title Controlled ocular drug delivery using peptide-mediated phase separation

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/20/2010

Defense Date 03/22/2010

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

Tag dry eye syndrome,elastin-like polypeptides,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Hamm-Alvarez, Sarah F. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email jashnani@usc.edu,jashnaniaarti@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2945

Unique identifier UC1198000

Identifier etd-Jashnani-3619 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-309284 (legacy record id),usctheses-m2945 (legacy record id)

Legacy Identifier etd-Jashnani-3619.pdf

Dmrecord 309284

Document Type Thesis

Rights Jashnani, Aarti

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu