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Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications

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Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications

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Content Expression and purification of different Elastin Like Polypeptides (ELPs) constructs for therapeutic applications by Tao Ma 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) August 2015 Copyright 2015 Tao Ma ! II! Table of Contents Acknowledgements List of Tables List of Figures Abbreviations Abstract 1. Introduction 1 1.1) Popularity of protein-based therapies 1 1.2) Phase transition behavior of ELPs 2 1.3) Factors that influence ELP transition temperature 4 1.4) Fermentation of ELP fusions in Escherichia coli 5 1.5) Removal of pyrogenic endotoxins from E. coli expressed proteins 6 1.6) Current treatments for dry eye disease 7 1.7) Lacritin, a promising therapeutic for dry eye disease 8 2. Materials and methods 11 2.1) Instruments and reagents 11 2.2) Transformation of LV96, V96 and S96 DNA into BLR cells 12 2.3) Expression and purification of LV96, V96 and S96 13 2.4) Scaling-up the purification of LV96 by Size Exclusion Chromatography (SEC) 14 2.5) Endotoxin removal and quantification of endotoxin burden 14 2.6) Protein lyophilization and quality control 15 ! III! 3. Results 16 3.1) Confirmation of DNA sequences for LV96, V96 and S96 16 3.2) Expression and purification of LV96, V96 and S96 19 3.3) Optimization of purification of LV96 20 3.4) Scaling-up the purification of LV96 from Superose 6 10/300 GL SEC column to HiLoad 26/600 Superdex 200 PG SEC column 22 3.5) Endotoxin removal from LV96, V96 and S96 24 3.6) Quantification of endotoxin level for LV96, V96 and S96 25 3.7) Protein lyophilization and quality control 26 4. Discussion 29 References 36 ! IV! Acknowledgements Studying at USC has been the best experience of my life, and I would like to take this opportunity to express my gratitude to all of those, who have helped me completed this thesis as well as in my master study. First and foremost, I would like to express my appreciation to my mentor, Dr. Sarah F. Hamm-Alvarez. I would like to thank her for giving me the opportunity to join her lab during my master study, from where I got the chance to involve in different fields of research, as well as her guidance and support. I also want to deeply thank Dr. J. Andrew MacKay, for allowing me to work on the lacritin project, from which I gained a lot of research skills. I would like to thank him for his patience and guidance, and allowing me to grow as an independent scientist. Additionally, I would like to thank Dr. Wei-Chiang Shen, for being one of the committee members, giving me feedbacks and suggestions on my master thesis. I also want to show my gratitude to Dr. Curtis T. Okamoto, for guiding me during my study here at USC as well as his encouragement and valuable suggestions. I would like to thank all lab members from both Dr. Sarah F. Hamm-Alvarez’s and Dr. J. Andrew MacKay’s lab, for teaching, guiding and helping me in my experiments. Lastly, I would like to thank my family and friends for their love and encouragement. I especially want to thank my parents for their unconditional love and support. ! V! List of Tables Table 1 Nomenclature, amino acid sequence, expected molecular weight and T t of proteins 13 Table 2 Optimization of purification of LV96 21 Table 3 Endotoxin removal from S96, V96 and LV96 using detoxi-gel column 25 Table 4 Endotoxin level quantification using the pyrogent assay 26 Table 5 Quality control for 10 µM 1 ml V96, S96 and LV96 using the pyrogent assay 28 ! VI! List of Figures Figure 1 Phase transition behavior of ELPs 3 Figure 2 Lacritin released from lacrimal gland acinar cells (LGACs) flows via ducts to the ocular surface 9 Figure 3 Cartoon showing the components of the LV96 fusion protein 10 Figure 4 DNA sequencing data for genes encoding LV96, V96 and S96 16 Figure 5 Expression and purification of V96 and S96 20 Figure 6 Optimization of purification of LV96 21 Figure 7 Scaling-up the purification of LV96 from Superose 6 10/300 GL SEC column to HiLoad 26/600 Superdex 200 PG SEC column 22 Figure 8 Endotoxin removal from S96, V96 and LV96 using detoxi-gel column 24 Figure 9 Quality control data for batches of V96, S96 and LV96 27 Figure10 Helical wheel shows the C-terminal amphipathic α-helix (NGSEFAQKLLKKFS) of lacritin 35 ! VII! Abbreviations ELPs, Elastin Like Polypeptides ITC, Inverse Transition Cycling IPTG, Isopropyl β-D-1-thiogalactopyranoside LPS, Lipopolysaccharide E. coli, Escherichia coli EU, Endotoxin Unit LCST, Lower Critical Solution Temperature T t , Transition Temperature SEC, Size Exclusion Chromatography DED, Dry Eye Disease SS, Sjögren’s syndrome SDC-1, Syndecan-1 SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis LGACs, Lacrimal Gland Acinar Cells PBS, Phosphate Buffered Saline V96, (VPGVG) 96 S96, (VPGSG) 96 LV96, Lacritin-(VPGVG) 96 ! VIII! Abstract There are many rationales for modifying recombinant proteins by using polymers, which include: i) altering permeability/diffusion via controlling their hydrodynamic radius; ii) modulating mediators of controlled release; and iii) improving their avidity to molecular/cellular targets. Many of these strategies require covalent bioconjugation, which are inefficient and may compromise protein activity. An emerging solution to this challenge is to develop gene products that act like polymers. One such polymer is known as the Elastin Like Polypeptides (ELPs). ELPs are ‘protein polymers’ composed of repetitive amino acid sequence derived from the human extracellular matrix protein called tropoelastin. In addition to their high molecular weight, which exceeds 80 kDa, ELPs undergo thermo-responsive phase separation. ELPs are highly soluble in aqueous solutions below their transition temperature (T t ) and rapidly self-assemble into viscous coacervate particles above T t . T t can be modulated by changing the molecular weight and hydrophobicity of the ELP. In this thesis, ELPs are developed as a carrier for lacritin, which is a novel tear glycoprotein that promotes tear secretion, maintains ocular surface integrity and reduces formation of lymphocytic foci in lacrimal gland. Free lacritin increases the basal tear secretion in rabbits; however, it must be administered frequently. The rationale for exploring fusions of lacritin and ELP is that they may prolong the retention on the anterior segment of the eye. In order to explore this strategy, my thesis has focused on scaling-up the production of high purity, low endotoxin level material for subsequent evaluation treating in models of aqueous-deficient dry eye diseases. E. coli was used for expression of the lacritin-ELP fusion construct and size exclusion chromatography (SEC) was used to separate LV96 from cleavage product. Endotoxin removal was explored using polymyxin chromatography. This thesis represents the first report of scaling-up production of low endotoxin level lacritin ELP fusions. ! 1! 1. Introduction 1.1) Popularity of protein-based therapies Protein-based therapies have shown robust growth in the global market in recent years, and it is expected to further increase 7%-15% annually (Walsh, 2010). Even though the approval of the first nucleic acid-based product in EU in 2012, Glybera TM , which was approved for treating lipoprotein lipase deficiency (LPLD) represents a milestone in the development of gene therapy, it seems that the majority of approvals over the next few years will continue to be protein-based products, for example, monoclonal antibodies (mAbs) (Walsh, 2014). The popularity of protein-based drugs lies in the fact that they are often highly specific in their biological interactions. In addition, there are an estimated thousands of human proteins that are still barely understood which may be modified to exert therapeutic effects according to genomics and bioinformatics data (Casteleijn et al., 2013). However, endogenous proteins face challenges in their delivery such as poor water solubility, weak physical or chemical stability, poor pharmacokinetic properties, and even cytotoxicity (Casteleijn et al., 2013; Du and Stenzel, 2014). To overcome these shortcomings, different protein carriers have been developed to enhance drug delivery. Generally, two classes of carriers have been developed: chemically synthesized carriers and genetically engineered carriers (Rabotyagova et al., 2011; Shi et al., 2014). Even though the clinical applications of genetically engineered carriers are still limited, they have certain unique properties that can be utilized in pharmaceutical research. For example, the ability to form micro- or nanostructures through interaction among well-defined protein secondary structures such as α-helices and β-sheets (Elemans et al., 2003; Koehl and Delarue, 1994; Shi et al., 2014). In addition, hierarchical structures offer genetically engineered carriers an increasing level of complexity, which exhibits more unique properties and functions (Elemans et al., 2003; Janib et al., 2014; Shi et al., 2014; ! 2! Sun et al., 2011). Thus, genetically engineered carriers are a promising approach for drug delivery. 1.2) Phase transition behavior of ELPs Elastin Like Polypeptides (ELPs) are one of the well-developed genetically engineered protein polymers, with which the endogenous proteins can be fused, to form recombinant proteins. Gene fusions are formed through splicing together genes or gene fragments to combine the characteristics of parental products (Uhlén et al., 1992; Uhlén and Moks, 1990). ELPs are inspired by human tropoelastin, which has two major domains. One of the two is hydrophobic domain rich in non-polar amino acids Gly, Val, Pro and Ala in the form of peptides repeats such as GVGVP, GGVP and GVGVAP. The other one is the hydrophilic domain rich in Lys and Ala in the form of separation of Lys by 2 or 3 Ala such as AAAKAAKAA, which is responsible for covalent cross-linking (Indik et al., 1989; Indik et al., 1987; Vrhovski and Weiss, 1998). Tropoelastin is soluble in aqueous solutions when the temperature is lower than 20°C while it forms coacervates as the temperature increases, resulting in the coacervation of tropoelastin molecules due to desolvation of structured water on hydrophobic chemical moieties. This process is thermodynamically controllable and reversible by lowering the temperature of solutions (Urry, 1988; Vrhovski and Weiss, 1998). Inspired by the repetitive hydrophobic domains of human tropoelastin, ELPs are biopolymers, which are composed of short repetitive pentapeptides (Val-Pro-Gly-Xaa-Gly) n , where the “guest residue” Xaa can be any amino acid residue and n is the number of the pentapeptide motifs (MacEwan and Chilkoti, 2014; Tatham and Shewry, 2000). ELPs exhibit lower critical solution temperature (LCST) phase transition behavior. LCST phenomena are commonly observed for synthetic polymers; ! 3! however, for ELPs this is often defined as a transition temperature (T t ). Similar to tropoelastin, ELPs are highly soluble in aqueous solution when the temperature is below the T t . They phase separate abruptly (<1°C range) when the temperature is raised above the T t , which leads to self-assembly into viscous coacervate particles. As the temperature decreases from above T t to below T t , coacervates reversibly solubilize in aqueous solution (Figure 1) (MacEwan and Chilkoti, 2014; Meyer and Chilkoti, 1999; Urry, 1988; Urry, 1992, 1997). This thermo-responsive phase transition property of ELPs gives drug a potential to exert controlled release through formation of drug depots at physiological temperatures by modulating T t . Due to this distinctive thermo-responsive and biocompatible properties, ELPs have been studied in different fields such as tissue engineering (Koria et al., 2011; Nettles et al., 2010), targeted cancer therapy (Callahan et al., 2012; MacKay et al., 2009) and in the development of stimuli responsive hydrogels (Wang et al., 2013a). Figure 1. Phase transition behavior of ELPs. T>T t , ELPs phase transition; T<T t , ELPs solubilize in aqueous solution. This process is thermodynamically controllable and reversible. Heat Cool ! 4! 1.3) Factors that influence ELP transition temperature Transition temperature is determined by the intrinsic parameters as well as extrinsic parameters of ELPs. Intrinsic parameters include the guest residue composition and the chain length of ELPs (molecular weight). The more hydrophobic the guest residue is, the lower the T t is (MacEwan and Chilkoti, 2014; McDaniel et al., 2013; Meyer and Chilkoti, 1999; Urry, 2004; Urry et al., 1991; Urry et al., 1985). The longer the chain length is (higher the molecular weight), the lower the T t shows (MacEwan and Chilkoti, 2014; Meyer and Chilkoti, 1999, 2004; Urry et al., 1985). In addition, several extrinsic parameters such as the concentration of ELPs solution, ionic strength and pH also influence T t for a certain ELP (MacEwan and Chilkoti, 2014; Meyer and Chilkoti, 1999; Urry, 1997). T t is inversely related to the concentration of ELPs, as greater ELPs concentrations exhibit lower T t (MacEwan and Chilkoti, 2014; Meyer and Chilkoti, 1999, 2004). This effect is significant when the concentration is less than 25 µM (Meyer and Chilkoti, 1999; VÉRtesy et al., 1984). The effects of salts that influence T t follow the Hofmeister series. Kosmotropic anions (Cl - and higher in Hofmeister series) depress T t , which is further enhanced by increasing salt concentration (Cho et al., 2008; MacEwan and Chilkoti, 2014). Furthermore, pH of the solution has an effect on the T t of ELPs that have ionizable guest residues (Callahan et al., 2012; MacEwan and Chilkoti, 2014; MacKay et al., 2010; Urry, 1997). This phase transition behavior enables the purification of ELPs through inverse transition cycling (ITC) according to their thermal response. During ITC, changing the temperature selectively precipitates and resolubilizes ELPs, which allows the removal of both soluble and insoluble impurities following protein expression in E. coli (MacEwan et al., 2014). Since the thermally controlled phase transition behavior of ELPs is maintained upon incorporation in a fusion protein (Chen and Huffman, 1990; Chilkoti et ! 5! al., 1994; Hoffman, 1987; Meyer and Chilkoti, 1999), ITC can also be used for purification of ELP fusions (MacEwan et al., 2014; Meyer and Chilkoti, 1999). 1.4) Fermentation of ELP fusions in Escherichia coli Recombinant DNA technology enables a predictable way to form in vitro gene fusions (Uhlén and Moks, 1990). The capability to express and purify recombinant proteins that possessing desired pharmacokinetics properties is required in the development of pharmaceutical proteins(Casteleijn et al., 2013; Papaneophytou and Kontopidis, 2014). Bacterial expression systems, particularly Escherichia coli (E. coli), together with mammalian and pichia expression system, have been used to produce large amount of proteins from cloned genes. As a commonly used expression system for industrial and pharmaceutical protein production, E. coli grows rapidly to high density on inexpensive substrates (Terpe, 2006). Function of this expression system is often based on the E. coli lac operon, which consists of three structural genes lacZ, lacY and lacA. Only lacZ and lacY appear to be necessary for lactose catabolism. lacY encodes the lactose permease which in mediates lactose uptake. lacZ encodes β-galactosidase, which cleaves lactose into glucose and galactose and produces a natural inducer of the lac operon, allolactose. The transcription of the lac operon relies on the repression by the lactose repressor LacI, which prevents transcription from the promoter itself by binding to the operator region of lac operon (Adhya, 1996; Deutscher et al., 2006; Marbach and Bettenbrock, 2012). Allolactose triggers transcription of the lac operon by binding to LacI, which releases LacI from the operator region. To achieve high expression of proteins, inducers of the lac operon such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) are widely used in molecular biology and biotechnology. As an analog of allolactose, IPTG also binds to LacI and releases ! 6! LacI from the operator region in an allosteric manner. Unlike allolactose, IPTG is not a substrate of β-galactosidase, and therefore, its concentration during experiment remains constant. Therefore, protein expression where the gene is under control of lac operator can be induced by IPTG to a significantly higher level (Hansen et al., 1998; Marbach and Bettenbrock, 2012). 1.5) Removal of pyrogenic endotoxins from E. coli expressed proteins One disadvantage of protein expression by utilizing E. coli is the accumulation of lipopolysaccharide (LPS), also generally referred as endotoxins, which show high toxicity in vivo and in vitro. The threshold level of endotoxins for intravenous injection is 0.25 endotoxin unit (EU)/ml (Petsch and Anspach, 2000; Terpe, 2006). Meeting this criterion has always been a challenge in pharmaceutical research since simple sterile filtration doesn't remove pyrogens to a great extent (Berthold and Walter, 1994; Petsch and Anspach, 2000). As one type of exogenous pyrogen, endotoxins are an integral part of the outer membrane of the cell wall of gram-negative bacteria. Structurally, endotoxins are composed of three different parts, which are a non-polar lipid component, lipid A; core oligosaccharide, which comprises inner core and outer core; and O-antigen (a heteropolysaccharide representing the surface antigen) (Ohno and Morrison, 1989; Petsch and Anspach, 2000). Endotoxins do not exert their toxicity by killing or inhibiting cellular functions of host cells. Instead, they elicit an active response from host cells (Rietschel et al., 1994). Activation of the immune system is initiated through the interactions between lipid A and various host cell types or organs, especially monocytes and macrophages. The activation of these cells results in the release of mediators, such as tumor necrosis factor (TNF), several interleukins (IL-1, IL-6, IL-8 and IL-10), colony stimulating factor, prostaglandins, platelet activating factor and free radicals, which are ! 7! capable of inducing endotoxin effects (Galanos et al., 1992; Galanos and Freudenberg, 1993; Petsch and Anspach, 2000; Rietschel et al., 1994; Vogel and Hogan, 1990). These are known to induce high fever, septic shock, and hypotension at high endotoxins exposure. Low levels of endotoxins might also contribute to several chronic diseases. In addition, increased blood endotoxin level leads to lung and kidney failure, intravascular coagulation and adult respiratory distress syndrome (Caroff and Karibian, 2003; Glauser et al., 1991; Lieder et al., 2013; Nalepka and Greenfield, 2004). Therefore, low levels of endotoxins are required in proteins that are expressed from gram-negative bacteria. 1.6) Current treatments for dry eye disease Dry eye disease (DED) is a common ocular disease in the US, which affects 17% of women and 11.1% of men (Moss et al., 2000). It is a multifactorial disease of tears and ocular surface causing discomfort, visual disturbance, tear film instability and further affecting the quality of life (Friedman, 2010; Listed, 2007). DED can be classified into two categories: aqueous-deficient dry eye disease with dysfunction of lacrimal gland and evaporative dry eye disease with dysfunction of meibomian gland (Javadi and Feizi, 2011; Qiao and Yan, 2013; Stern et al., 1998). As one group of aqueous-deficient DED, Sjögren’s syndrome (SS) is the second most common autoimmune disease in the US, which is characterized by inflammation of lacrimal gland and salivary gland (Akpek et al., 2011; Hessen and Akpek, 2014). Currently, the recommended treatments for SS-associated dry eye include topical administration of lubricants, inflammation inhibition strategy, and tear-conserving approaches (Akpek et al., 2011). However, none of these methods are satisfactory enough to provide the regulatory functions that maintained by the endogenous regulatory proteins found in normal tears (Karnati et al., ! 8! 2013; McKown et al., 2009; Wang et al., 2015). Thus, delivery of regulatory tear proteins is a promising approach to treat DED and SS (Wang et al., 2015). 1.7) Lacritin, a promising therapeutic for dry eye disease As a novel identified secreted tear glycoprotein, lacritin has a molecular weight of 12.3kDa. It exhibits lacrimal gland-specific expression at both the mRNA and protein levels. Lacritin is a lacrimal functional unit (LFU)- specific growth factor that is mainly generated by the lacrimal gland and apically released through acinar cell secretory granules, then transits through ducts to target corneal epithelial cells onto the ocular surface (Figure 2) (Ma et al., 2008; Sanghi et al., 2001). Studies have shown that lacritin is one of the only 4-5% of tear proteins that are down-regulated in dry eye or dry eye related syndrome (Green-Church et al., 2007; Kitagawa et al., 2007; Koo et al., 2005; McKown et al., 2009). As a cell selective mitogenic protein, lacritin promotes epithelial proliferation of HCE and human embryonic kidney cells driven by C-terminal domain (Wang et al., 2006). This mitogenic signaling requires the heparanase deglycanation of one cell surface protein, syndecan-1 (SDC-1), which exposes the lacritin binding site on N-terminus (Ma et al., 2006). Binding of the C-terminal domain of lacritin with N-terminal domain of SDC-1 triggers rapid signaling to downstream NFAT and mTOR pathways (McKown et al., 2009). Lacritin also promotes basal tear secretion by New Zealand White adult female rabbit (Samudre et al., 2011) and Aire KO mice (a model of SS disease) via topical treatment (Vijmasi et al., 2014). In addition, lacritin rapidly restores homeostatsis to stressed epithelial cells that have been stressed by inflammatory interferon-γ and tumor necrosis factor through acceleration of autophagy (Wang et al., 2013b). Moreover, topical lacritin maintains ocular surface integrity (Vijmasi et al., 2014; Wang et al., 2014a) and suppresses the lymphocytic foci formation in the lacrimal glands ! 9! of Aire KO mice (Vijmasi et al., 2014). Thus, the discovery of lacritin offers a new potential therapeutic for DED (McKown et al., 2009). Figure 2. Lacritin released from lacrimal gland acinar cells (LGACs) flows via ducts to the ocular surface (Wang et al., 2006). Our group generated a novel lacritin-ELP fusion construct (LV96), which maintains the thermo-responsive property of the parent ELP (V96) and the efficacy of lacritin (Figure 3). Our group has shown that LV96 maintains the prosecretory efficacy of lacritin, as demonstrated by its capability to dose-dependently stimulate β-hexosaminidase secretion from primary rabbit LGACs. In addition, LV96 increased tear secretion from female non-obese diabetic (NOD) mice (a model of SS disease) upon intra-lacrimal injection. This treatment has been found to produce a drug depot in LG. Moreover, intracellular trafficking and transcytosis of exogenous lacritin in LGACs was prolonged by fusion to V96 (Wang et al., 2015). In addition to using V96 as the backbone ELP for lacritin, a diblock ELP (SI) nanoparticle scaffold has been previously reported to fuse to lacritin. This fusion protein is called LSI, which showed enhancement of cellular uptake, Ca 2+ mediated signaling and scratch closure in a human corneal epithelial cell line. Moreover, topical administration of LSI onto the ocular surface of female NOD mice (a model for impaired wound healing in human) accelerated the recovery of the corneal epithelium ! 10! (Wang et al., 2014a). Therefore, fusion to ELP nanoparticles appears to be a new strategy to more efficiently deliver lacritin to exert its biological function. Figure 3. Cartoon showing the components of the LV96 fusion protein. In our collaborator’s previous study, New Zealand White adult female rabbit (3-5kg) with normal eyes was used as the model for studying basal tearing by lacritin. In the study, lacritin was reported to acutely increase basal tear secretion for at least 240 mins after a single dose (50µg/ml). Moreover, eyes treated with lacritin three times per day for two weeks showed a steady increase in tearing, which was continually sustained for at least another one week after the last treatment and was well tolerated (Samudre et al., 2011). However, three times daily administration is inconvenient for patients. To potentially reduce the frequency of topical administration, this collaborative study is focusing on modifying lacritin to possess controlled release property, which will reduce the frequency of administration. In this study, ELP (V96) was developed as a drug carrier to deliver lacritin so that a drug depot will form under the physiological condition and may be able to prolong the lacritin retention on the anterior segment of the eye. Basal tear secretion will be examined after topically administering lacritin-ELP fusion protein (LV96) one dose per day onto the ocular surface of the same rabbit model. This study highlights the potential of ELP nanoparticles as controlled release drug carriers for delivering regulatory proteins found in normal tears. ! Lacritin Fusion!site ELP!tag ! 11! For this master’s thesis, two plain ELP carriers (V96 and S96) and one lacritin-ELP fusion construct (LV96) were expressed in E. coli. Several rounds of ITC were used to purify these proteins. One challenge for purification is the stability of LV96. ELP spontaneously cleaves from fusion construct, which leads to a combination of fusion protein and free ELP after ITC. Also, native lacritin has a cleavage half-life of about one day at 37°C (Wang et al., 2015). Thus, size exclusion chromatography (SEC) was used for purification of LV96. In addition, 5-fold scaling up of the loading amount of LV96 onto SEC columns was achieved by shifting from using a small SEC column to a big one. Moreover, endotoxins were removed from protein samples by using detoxi-gel columns before lyophilization. This work shows the first time scaling-up of the low endotoxin level LV96 in our lab. Finally, protein powder was re-suspended, purity was confirmed by running samples onto SDS-PAGE gel, T t was measured, and the endotoxin level was confirmed as quality control. Then samples were shipped on dry ice to collaborators in Virginia for further in vivo study. 2. Materials and methods 2.1) Instruments and reagents Terrific broth dry powder growth medium was purchased from MO BIO Laboratories, Inc. (Carlsbad, CA). Isopropyl β-D-1-thiogalactopyranoside, OmniPur*.99.0% min. was purchased from VWR (Visalia, CA). Top 10 competent cells were purchased from Invitrogen (Rockford, IL). BLR (DE3) competent cells were purchased from Novagen (Milwaukee, WI). Amicon Ultra concentrators were purchased from Millipore (Billerica, MA). 4-20% Tris-Glycine PAGEr gels were purchased from LONZA (Allendale, NJ). ! 12! QIAprep spin Miniprep kit was purchased from Qiagen (Valencia, CA). Superose 6 10/300 GL SEC column was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). HiLoad 26/600 Superdex 200 prep grade SEC column was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). Detoxi-Gel TM Endotoxin Removal Columns were purchased from Thermo Fisher Scientific (Rockford, IL). Pyrogent TM Plus Gel Clot LAL single test vials were purchased from LONZA (Allendale, NJ). 2.2) Transformation of LV96, V96 and S96 DNA into BLR cells The sequences encoding LV96, V96 and S96 were synthesized in our lab and stored in a DNA library. The amino acid sequences of ELPs used in this study are described in Table 1. Transformation of LV96, V96 and S96 plasmid DNA into Top10 cells were performed for DNA replication. Purified plasmid DNA were obtained by using QIAprep spin Miniprep kit, then the genes for LV96, V96 and S96 were confirmed by DNA sequencing. Then transformed LV96, V96 and S96 DNA were transfected into BLR (DE3) E. coli. for protein expression. However, the gene for V96 could not be confirmed by examining the sequencing alone, so diagnostic digestion was carried out to further confirm the sequence by using BseRI and BamHI to cut the V96 plasmid DNA. ! 13! Protein Label Amino Acid Sequence Expected M.W. (kDa) Tt (°C) V96 G(VPGVG) 96 Y 39.55 31.6 S96 G(VPGSG) 96 Y 38.39 55.0 LV96 GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQE TSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKG MHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPRGSG( VPGVG) 96 Y 52.52 26.8 Lacritin GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTAQE TSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKG MHGGVPGGKQFIENGSEFAQKLLKKFSLLKPWAGLVPR 12.84 NA Table 1. Nomenclature, amino acid sequence, expected molecular weight and T t of proteins 2.3) Expression and purification of LV96, V96 and S96 V96 and S96 were expressed in BLR E. coli by shaking in an orbital shaker at 37°C for 24h at 250 rpm. 500 µl 1M IPTG was added into 1L TB medium during LV96 expression. Expressed LV96 for around 5h at 37°C and measured OD value for the medium at 600 nm wavelength at certain time points (every 30 mins) during this expression, until the value reached 0.5. Then added IPTG into the medium and immediately lowered the temperature from 37°C to room temperature and continued expressing for another 6h. Cell cultures were harvested and re-suspended in phosphate buffer saline (PBS). Then inverse transition cycling (ITC) was performed for protein purification. To do this, the temperature was increased to 37°C followed by hot spin (centrifugation at 37°C), then ELPs phase separated and precipitated as pellets which were then collected. Then the pellets were re-suspended in PBS and the temperature was decreased to 4°C followed by cold spin (centrifugation at 4°C). ELPs were solubilized in the supernatant after ! 14! centrifugation, which was then collected. 5 rounds of ITC were performed to purify V96 and S96. 3 rounds of ITC were optimized to purify LV96. The purity of ELPs were determined by SDS-PAGE gels stained with CuCl 2 or Coomassie blue. Protein concentrations were determined by using Nanodrop 2000 spectrophotometer at 280nm (εV96=1280 M -1 cm -1 , εS96=1280 M -1 cm -1 , εLV96=6970 M -1 cm -1 ). 2.4) Scaling-up the purification of LV96 by Size Exclusion Chromatography (SEC) Since LV96 is partially proteolyzed during biosynthesis, fusion proteins were further purified by using a Superose 6 10/300 GL size exclusion column, which has a column volume of 24 ml, at 4°C. 1 ml of LV96 sample with the concentration of 500 µM was loaded onto the column and washed out by isocratic flow of PBS at 0.5 ml/min. To scale up the production of pure LV96, a HiLoad 26/600 Superdex 200 prep grade size exclusion column was used, which has a column volume of 330 ml, at 4°C. 5 ml of LV96 sample with the concentration of 500 µM was loaded onto the column and washed out by isocratic flow of PBS at 2.6 ml/min. For both columns, the first peak that showed on chromatograms represents LV96, which was then collected and concentrated by an Amicon Ultra concentrator (10KD cut off) at 4°C. 2.5) Endotoxin removal and quantification of endotoxin burden 700 µM, 300 µl of LV96, V96 and S96 were loaded onto the Detoxi-Gel TM Endotoxin Removal Columns, respectively, after column regeneration. S96 was incubated in the column for 1h at room temperature. LV96 and V96 were incubated in columns for 1h on ! 15! ice. After incubation, the columns were washed out by cell culture PBS then elution was collected in tubes with 250 µl elution per tube. The collected elution tubes that had the highest concentration for each protein were selected and they were subsequently diluted 10 2 , 10 3 , 10 4 , 10 5 , 10 6 -folds. Then endotoxin level was quantified by incubating 250 µl of each dilution in Pyrogent TM Plus Gel Clot LAL single test vials covered by aluminum foil at 37°C for 1h. 2.6) Protein lyophilization and quality control The collected elution for each protein were combined and diluted into 10 µM. Then diluted samples were then aliquoted into 25 different tubes, each containing 1 ml protein. Samples were lyophilized for 24h and tubes were wrapped with parafilm in case of contamination of endotoxins. Powder was re-suspended by using 1 ml cell culture PBS for V96 and S96 (V96 and S96 were lyophilized out of endotoxin-free water) and 1 ml endotoxin-free water for LV96 (LV96 was lyophilized out of cell culture PBS) and then quality control was performed. Firstly, 12 µg of LV96, V96 and S96 were loaded onto SDS-PAGE gels followed by Coomassie blue staining. Then transition temperature was measured using DU800 UV-Vis spectrophotometer for LV96, V96 at 10 µM, S96 at both 10 µM and 25 µM. Finally, 250 µl V96 and S96 were used and 10 2 , 10 3 -fold LV96 dilutions were used to carry out pyrogent assay to quantify the endotoxin level. Then samples were shipped to collaborators in Virginia on dry ice for further in vivo study. ! 16! 3. Results 3.1) Confirmation of DNA sequences for LV96, V96 and S96 DNA sequences for LV96, V96 and S96 were confirmed (Figure 4). The sequences for LV96 and S96 were confirmed the same as in the literature (Figure 4A &B). However, there were undetermined amino acids in the sequence of V96 (Figure 4C). So I looked into the sequencing chromatogram of V96 and changed the N to G on the 74 th position, then the sequence was confirmed (Figure 4D). In order to further confirm the sequence for V96, diagnostic digestion was performed by using BseRI and BamHI cutting the plasmid DNA, which yielded a band around 1.5 kilo bases, which was accorded with the theoretical value of 1486 nucleotides (Figure 4E). A ! 17! B C ! 18! D E ! 19! Figure 4. DNA sequencing data for genes encoding LV96, V96 and S96. A) LV96 forward and reverse sequence. B) S96 forward and reverse sequence. C) V96 forward and reverse sequence. D) Undetermined amino acids in the sequence of V96. Changed the N to G on the 74 th position on sequencing chromatogram yielded the right sequence for V96. E) Diagnostic digestion for V96 by using BseRI and BamHI cutting V96 plasmid DNA, which yielded a band around 1.5 kilo bases. 3.2) Expression and purification of LV96, V96 and S96 V96 and S96 were expressed in E. coli and purified by 5 rounds of ITC. Purities were confirmed by loading samples onto an SDS-PAGE gel followed by CuCl 2 staining. V96 single band was seen on the gel. However, single band could not be seen on the gel for S96. Since the T t for S96 is 55°C, it is not easy for protein to phase separate, so higher amounts of NaCl were added to facilitate lower the T t during protein purification. However, impurities were also precipitated in addition to phase separation of S96 (Figure 5). ! 20! Figure 5. Expression and purification of V96 and S96. SDS-PAGE gel for purified V96 and S96 (10 µg and 25 µg). Gel was stained using CuCl 2 . V96 single band was seen on the gel, while S96 single band couldn't be obtained. 3.3) Optimization of purification of LV96 LV96 was also expressed in E. coli and purified by ITC. Purity was confirmed by loading samples, which were collected after each round of ITC, onto an SDS-PAGE gel followed by CuCl 2 staining. Single band couldn't be obtained even after 6 rounds of ITC since LV96 keeps cleaving on the fusion site between lacritin and V96 (Figure 6A). Since more rounds of ITC give more chances for LV96 to cleave, optimization of purification was performed by analyzing the intensity of LV96 as a function of ITC with Image J. The intensity of LV96 didn't further increase after 3 rounds of ITC, suggesting that 3 rounds of ITC were sufficient for purification of LV96 (Table 2). To further confirm this ! 21! optimization, 3 rounds of ITC were performed for purification of LV96 and purity was confirmed by loading samples onto an SDS-PAGE gel followed by CuCl 2 staining again. This gel showed less free ELP (V96) intensity (Figure 6B). A B Figure 6. Optimization of purification of LV96. A) SDS-PAGE gel for purified LV96 (10 µg) after each round of ITC. Gel was stained using CuCl 2 . Lane 2 to lane 7 represent LV96 sample after each round of ITC from first round to 6 th round from the same tube. Lane 8 to lane 12 represent LV96 after 6 rounds of ITC from 5 other different tubes. B) Confirmation of optimization of purification of LV96. SDS-PAGE gel for purified LV96 (10 µg) after 3 rounds of ITC. ITC 1 ITC 2 ITC 3 ITC 4 ITC 5 ITC 6 Intensity of LV96 42.9% 39.0% 50.2% 54.2% 45.8% 51.9% Table 2. Optimization of purification of LV96. Analyzed the intensity of LV96 as a function of ITC by using Image J. 3 rounds of ITC were sufficient for purification of LV96. ! 22! 3.4) Scaling-up the purification of LV96 from Superose 6 10/300 GL SEC column to HiLoad 26/600 Superdex 200 PG SEC column In order to separate LV96 from free ELP (V96) after 3 rounds of ITC, LV96 was loaded onto the SEC columns. Firstly, 1 ml of LV96 sample with concentration of 500 µM was loaded onto the Superose 6 10/300 GL (small) SEC column. Chromatogram showed the separation of LV96, which was represented by the first peak, from V96. LV96 started to be washed out at around 14 min (Figure 7A). Then 5 ml LV96 sample with concentration of 500 µM was loaded onto the HiLoad 26/600 Superdex 200 PG (big) SEC column. The separation of LV96, which was also represented by the first peak, from V96 was observed. LV96 started to be washed out at around 36 min (Figure 7B). Then 30 µg of sample from peak 1 and peak 2 were loaded onto SDS-PAGE gel to see the purity of LV96 after SEC column followed by Coomassie blue staining. Relatively pure LV96 (peak 1) were obtained after SEC columns (Figure 7C). A ! 23! B C Figure 7. Scaling-up the purification of LV96 from Superose 6 10/300 GL SEC column to HiLoad 26/600 Superdex 200 PG SEC column. A) Chromatogram after loading LV96 onto Superose 6 10/300 GL SEC column. B) Chromatogram after loading LV96 onto HiLoad 26/600 Superdex 200 PG SEC column. C) SDS-PAGE gel for peak 1 and peak 2 (30 µg) after SEC column. Gel was stained by Coomassie blue. ! 24! 3.5) Endotoxin removal from LV96, V96 and S96 Proteins were loaded onto detoxi-gel columns and then incubated for 1h. The absorbances for elution were measured at 280 nm and 350 nm wavelength and then A280-A350 versus elution volume were plotted. S96 was incubated at room temperature for 1h and then washed out by cell culture PBS, with 250 µl elution per tube, 8 tubes in total. The highest concentration was 445.3 µM from elution tube 4 (Figure 8A, Table 3). V96 was incubated on ice for 1h and then washed out by cell culture PBS, with 250 µl elution per tube, 9 tubes in total. The highest concentration was 567 µM from elution tube 5 (Figure 8B, Table 3). LV96 was incubated on ice for 1h and then washed out by cell culture PBS, with 250 µl elution per tube, 5 tubes in total. The highest concentration was 182.2 µM from elution tube 2 (Figure 8C, Table 3). ! Figure 8. Endotoxin removal from S96, V96 and LV96 using detoxi-gel column. A) A280-A350 versus elution volume (µl) for S96. B) A280-A350 versus elution volume (µl) for V96. C) A280-A350 versus elution volume (µl) for LV96. ! 25! ! Concentration (µM)( Tubes(of(Elution( S96( V96( LV96( Tube(1( 0! 0! 2.9! Tube(2( 0! 82.5! 182.2! Tube(3( 234.4! 364.5! 48.4! Tube(4( 445.3! 502.2! 4.3! Tube(5( 414.1! 567! 0! Tube(6( 203.1! 285! ! Tube(7( 46.9! 133.5! ! Tube(8( 0! 102! ! Tube(9( ! 19.5! ! Table 3. Endotoxin removal from S96, V96 and LV96 using detoxi-gel column. Concentration of protein elution in each tube, 250 µl elution per tube. ! 3.6) Quantification of endotoxin level for LV96, V96 and S96 The elution with the highest concentration for each protein were used to perform pyrogent assay. S96 elution from tube 4 was diluted 100-fold, and then showed negative result, which suggested that the endotoxin level was lower than 6 EU/ml. V96 elution from tube 5 was also diluted 100-fold. Result was also negative, which suggested that the endotoxin level was lower than 6 EU/ml. However, endotoxin level for LV96 elution from tube 2 couldn't decrease as much as S96 or V96 did. So a series of dilutions were carried out to quantify the endotoxin levels for LV96 elution. Result showed negative after 10 5 -fold dilution, which suggested that the endotoxin level was lower than 6000 EU/ml. To further confirm that the detoxi-gel column was effective in removing ! 26! endotoxins in some degree for LV96, 180! µM (the same concentration as LV96 elution from tube 2) LV96 sample without passing over the detoxi-gel column showed negative result when diluted to 10 6 -fold (Table 4). Results of pyrogent assay S96 (445.3 µM) V96 (567 µM) LV96 (182.2 µM) LV96 before column (180 µM) 1:10 2 dilution Negative Negative Positive Positive 1:10 3 dilution Positive Positive 1:10 4 dilution Positive Positive 1:10 5 dilution Negative Positive 1:10 6 dilution Negative Table 4. Endotoxin level quantification using the pyrogent assay. Endotoxin level were lower than 6 EU/ml for S96 and V96. However, endotoxin level was lower than 6000 EU/ml for LV96. Before detoxi-gel column, endotoxin level for LV96 was lower than 60000 EU/ml. 3.7) Protein lyophilization and quality control The elution for V96, S96 and LV96 were combined, respectively. The final concentrations for each protein are 770.4 µM for V96, 272.3 µM for S96 and 159.2 µM for LV96. Then each protein was diluted to 10 µM, and aliquoted into 25 tubes, with 1 ml in each tube, followed by lyophilization. The obtained protein powder was resuspended ! 27! in 1 ml cell culture PBS (for V96 and S96) or 1 ml endotoxin-free water (for LV96) and quality control was performed. Firstly, 12 µg of each sample was loaded onto SDS-PAGE gel. Clearly, there was a single band for each protein after Coomassie blue staining (Figure 9A). Then, transition temperature was measured for each protein at 10 µM. Since S96 didn’t have a good transition profile at low concentration, T t was further measured at 25 µM for S96. Thus, T t at 10 µM for V96 was 30.3°C, S96 was 61.2°C and LV96 was 26°C, which accorded with the literature (Figure 9B). Finally, endotoxin levels were further quantified for these proteins. V96 and S96 were re-suspended in 1 ml cell culture PBS and the result of pyrogent assay was negative, which suggested that endotoxin level were lower than 0.06 EU/ml. LV96 was re-suspended in 1 ml endotoxin-free water, pyrogent assay was also negative after sample was diluted to 10 3 -fold, which suggested that the endotoxin level was lower than 60 EU/ml. Result of pyrogent assay was negative for 10 µM LV96 sample that without passing over detoxi-gel column after 10 4 -fold dilution, which suggested that the endotoxin level was lower than 600 EU/ml (Table 5). A ! 28! B Figure 9. Quality control data for batches of V96, S96 and LV96. A) SDS-PAGE gel for V96, S96 and LV96. Single band showed for each protein on the gel. Gel was stained using Coomassie Blue. B) Transition temperature for V96 was 30.3°C, S96 was 61.2°C and LV96 was 26°C at 10 µM. Results for pyrogent assay (quality control) S96 (10 µM) V96 (10 µM) LV96 (10 µM) LV96 before column (10 µM) No dilution Negative Negative Positive Positive 1:10 2 dilution Positive Positive 1:10 3 dilution Negative Positive 1:10 4 dilution Negative Table 5. Quality control for 10 µM 1 ml V96, S96 and LV96 using the pyrogent assay. Endotoxin level for V96 and S96 were lower than 0.06 EU/ml. Endotoxin level for LV96 was lower than 60 EU/ml. Endotoxin level for 10 µM LV96 before detoxi-gel column was lower than 600 EU/ml. ! 29! 4. Discussion Protein-based drugs play an important role as therapeutics in treating oncology, diabetes, as well as cardiovascular, and infectious diseases (Du and Stenzel, 2014). Challenges in the administration of protein-based drugs include low hydrolytic stability, off-target side effects, rapid clearance from circulation and cytotoxicity (Du and Stenzel, 2014; Shi et al., 2014). To overcome these limitations in drug administration, multifunctional nanocarriers such as liposomes, carbon nanotubes and polymeric nanocarriers have been developed to assist drug delivery (Pérez-Herrero and Fernández-Medarde, 2015). As an emerging tool, genetically engineered carriers have been developed to address the above-mentioned challenges since they are precisely controlled in structure and size. Additionally, their specific biodegradable profile and fully customizable properties can be further tailored for specific applications in pharmaceutical research (Shi et al., 2014). Together with elastin-like polypeptides (ELPs), examples of well-studied genetically engineered drug carriers include silk-like polypeptides (SLPs), silk-elastin-like polypeptides (SELPs) and extended recombinant polypeptide (XTEN) polymers (Shi et al., 2014). One advantage of protein polymers is that different hydrophobicity, charges or secondary structures can be created through changing several amino acids sequences in pharmaceutical research. As one of the well developed genetically engineered polymeric drug carriers, sequences of ELPs can be precisely controlled, like the other protein polymers. (Cappello et al., 1990; Frandsen and Ghandehari, 2012; Shah et al., 2012; Shi et al., 2014; Urry et al., 2010). Treatment of ocular diseases has always been a challenge due to the difficulties in overcoming the eye-associated barriers, such as precorneal tear film, corneal barrier and conjunctival barrier. Moreover, poor physicochemical property of drugs is another factor ! 30! that hinders the ocular drug delivery. Only drugs with low molecular weight and moderate lipophilicity can bypass these barriers in a moderate manner. In order to facilitate delivery of drugs, besides chemical modification, suitable drug carriers have been developed to increase drug bioavailability (Reimondez-Troitiño et al., 2015). Nanotechnology-based formulation is one of the approaches. Ocular delivery nanocarriers have shown to: i) incorporate a large variety of drugs, ii) prolong the drug residence time on ocular surface and reduce drug degradation, iii) improve bioavailability via improving interactions between drugs and corneal epithelial (De la Fuente et al., 2010; Paolicelli et al., 2009; Reimondez-Troitiño et al., 2015; Singh and Jones, 2014; Souza et al., 2014). Nowadays, nanotherapies have been developed in treating ocular infections and inflammation, dry eye syndrome, glaucoma and for targeting the posterior segment of the eye (Reimondez-Troitiño et al., 2015). Sjögren’s syndrome (SS) is the second most common autoimmune disease in the US and it is characterized by inflammation of lacrimal gland and salivary gland, which leads to the symptoms of dry eye and dry mouth. As a secreted tear glycoprotein, lacritin has shown the potential to treat aqueous-deficient dry eye disease since it promotes basal tear secretion (Samudre et al., 2011; Vijmasi et al., 2014), maintains ocular surface integrity (Vijmasi et al., 2014; Wang et al., 2014a) and reduces the formation of lymphocytic foci in lacrimal gland (Vijmasi et al., 2014). However, the mechanism of how lacritin works to treat dry eye disease after topical administration has not been addressed. Since lacritin secrets from LGACs then flows via ducts to the ocular surface under physiological condition, one of the possible mechanisms of lacritin treating dry eye disease might be travel up the ducts and then go back to lacrimal gland after topical administration, which needs to be further confirmed in future study. ! 31! In this study, two free ELPs and one fusion ELP were expressed and purified. The guest residue for both LV96 and V96 is valine and the number of pentapeptide repeat is 96. Since the T t for V96 is around 31°C, which belows physiological temperature of 37°C, a drug depot that has controlled release property will form under the physiological condition. In LV96 fusion construct, V96 was chosen as the ELP backbone since T t for LV96 is even lower, which is around 26°C (Wang et al., 2015). The guest residue for S96 is serine and the number of pentapeptide repeats is also 96. In research, S96 is always used as a carrier control that doesn't undergo thermo-responsive self-assembly under physiological condition since T t for S96 is around 55°C (Wang et al., 2014d). This higher transition temperature also gives a challenge in purification process since higher amounts of NaCl were added to lower the T t during ITC in order to facilitate the phase transition of S96. However, it also precipitated impurities as shown on SDS-PAGE gel even after 5 rounds of ITC. The fusion protein LV96 confers the characteristics of both lacritin and V96: i) maintains the efficacy of lacritin, ii) forms a vicious drug depot at physiological temperatures to achieve controlled release property. Fusion lacritin protein exhibits a similar phase separation property with a decrease of T t about 5°C compared to the free V96 tag. After 6 rounds of ITCs, a single band of LV96 still couldn't be observed. SDS-PAGE analysis of purified LV96 suggested that a spontaneous cleavage of V96 from LV96 fusion construct. In addition, lacritin has a cleavage half-life of about 24h at 37°C (Wang et al., 2015). Serine protease may be responsible for the cleavage of lacritin next to the lysine residues, which further liberates an amphipathic α-helix on C-terminal for SDC-1 binding. Lacritin binding of SDC-1 triggers promitogenic signaling involves Gαi or Gαo–PKCα-PLC– Ca 2+ –calcineurin–NFATC1 and Gαi or Gαo–PKCα-PLC–phospholipase D (PLD)–mTOR pathways (McKown et al., 2009). Therefore, in order to maintain a single band on ! 32! SDS-PAGE gel for LV96, samples were kept on ice/ at low temperatures all the time and all experimental procedures were carried out as fast as possible. To reduce the handling time during purification, ITC was optimized to 3 rounds. In addition to ITC, size exclusion chromatography (SEC) was used at 4°C to separate LV96 from free V96. Two SEC columns were used, started from using a small column with the loading amount of about 26 µg. Then a big column with the loading amount of about 130 µg was used for scaling up. This is the first time a high volume column was used in our laboratory for purification of proteins. The loading amount of protein increased about 5-fold compared to the small column, which enables a more efficient approach for protein purification. Thus, more applications of SEC column may be applied in purifying ELPs in the future. SEC column may be used as an alternative strategy for ELPs purification. Technically, changing the temperature of the column, which is loaded with ELPs sample, changes the state of ELPs. Then washing out the column at different temperatures enables to separate ELPs from impurities. Firstly, load protein samples onto the column at low temperature. Then increase the temperature of the column to above the T t to initiate phase transition of ELPs. In this case, ELPs will attach to the column. Then wash out the column at higher temperature enables the elution of impurities. Subsequently, lower the temperature of the column to below the T t to re-solubilize ELPs. And then eluting the column again enables us to obtain relatively pure ELPs. In this way, protein purification can be more efficient and ready to scale-up for industrial production. There are two kinds of bacterial toxins: endotoxin and exotoxin. Endotoxin contamination is one major disadvantage of protein expression in E. coli. As an integral part of the cell wall of gram-negative bacteria, endotoxins are released on bacterial death ! 33! and in part during growth. The level of endotoxins should be maintained at low level for proteins that expressed from gram-negative bacteria due to their toxicity. Even though one study has shown that ITC is an effective method for removal of endotoxin for certain ELP polymers (McHale et al., 2005), however, it was not sufficient for the proteins in this study. Different endotoxin-selective ligands have been developed for endotoxin removal, such as polymyxin B, DEAE, histidine and Poly (L-lysine) (Petsch and Anspach, 2000). For removing endotoxin from ELPs, polymyxin (Schaal, 2012; Solomon et al., 2004) and ReductEtox resin have been reported to reduce the endotoxin level (Hrabchak et al., 2010). In this study, the Detoxi-Gel TM Endotoxin Removing Gel uses immobilized polymyxin B to bind and remove endotoxin from protein solutions. Polymyxin is a family of antibiotics that contain a cationic cyclopeptide with a fatty acid chain. Polymyxin B neutralizes the activity of endotoxins by binding to the lipid A portion of lipopolysaccharide, which further deorganizes the bacterial wall after insertion and breaks down endotoxin aggregates (Lopes and Inniss, 1969; Newton, 1956; Petsch and Anspach, 2000). Endotoxin removals were performed at room temperature and on ice for S96 and V96, respectively. Both of the proteins showed relatively low endotoxin levels after flowing through the detoxi-gel column. However, endotoxin removal on ice for LV96 was not so efficient as expected, which accorded with the finding that immobilized polymyxin B inactivates some but not all endotoxins (Kluger et al., 1985).There was no protein washed out when incubated sample twice by using detoxi-gel column, which further conformed to the finding that protein losses during passage through polymyxin B columns (Anspach and Hilbeck, 1995; Karplus et al., 1987). In addition, results of endotoxin removal vary from batch to batch for LV96. Thus, there is a need to investigate the differences in endotoxin removal between LV96 and the other two proteins. ! 34! Lacritin-deletion analysis identified a C-terminal mitogenic domain with an amphipathic α-helical structure (Wang et al., 2006), which is common to ligand-receptor or ligand-ligand interactions (Barden et al., 1997; Siemeister et al., 1998). The C-terminal sequence NGSEFAQKLLKKFS is essential for mitogenesis and NGSEFAQKLL is the active region. In addition, hydrophobic surfaces on the same or different proteins favors the formation of α-helical (Murre et al., 1989). From “helical wheel”, half of the residues in the active region are hydrophobic (A, F, G, L) and grouped to one face in the form of an amphipathic α-helix, which forms a hydrophobic-binding face (Figure 10) (Wang et al., 2006). Generally, amphipathic α-helices is one of the four prominent structures for antimicrobial peptides (Boman, 1995; Hancock, 1997; Jenssen et al., 2006). The interactions between antibacterial peptides with lipopolysaccharides are driven by electrostatic and hydrophobic interactions. The electrostatic interaction occurs through electrostatic bonds between cationic peptides and negatively charged phosphate groups within LPS. Additionally, there is a hydrophobic interaction between hydrophobic domain of the amphipathic peptides and hydrophobic LPS component, lipid A (Jenssen et al., 2006). These kinds of interactions might also stabilize the binding between LV96 and LPS, which could be the reasons that account for the low efficiency of endotoxin removal for LV96. Since lacritin possesses a C-terminal amphipathic α-helix, the hydrophobic domain may interact with lipid A portion within LPS. While the hydrophilic domain, which possesses positive charge, may interact with the negatively charged LPS. These may be the two interactions that stabilize LV96 with LPS. 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Abstract (if available)

Abstract There are many rationales for modifying recombinant proteins by using polymers, which include: i) altering permeability/diffusion via controlling their hydrodynamic radius

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Creator Ma, Tao (author)

Core Title Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/14/2015

Defense Date 06/09/2015

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

Tag elastin like polypeptides,ELPs,endotoxin removal,lacritin,OAI-PMH Harvest,protein expression,scaling-up of protein purification

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hamm-Alvarez, Sarah F. (committee chair), Mackay, John Andrew (committee member), Shen, Wei-Chiang (committee member)

Creator Email matao8901@163.com,taom@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-592829

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Legacy Identifier etd-MaTao-3594.pdf

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