<p> Proteins vary tremendously in many of their physical and chemical properties. In order to perform in vitro application or analysis, one protein must be separated from other cellular components. This process is called protein purification. With the advances of modem science and technology, many protein purification schemes have been developed. Among them, the ELP-intein protein purification system has recently attracted an increasing amount of attention because of its positive characteristics: it is simple, inexpensive, scalable, with a high throughput, protease-free, etc. However, although the scientific literature reports all those good aspects of the system, several bad responses to it still exist. In this thesis, through comparing expression and purification of four different self-cleavage ELP fusion proteins, we propose a general solution to these problems for the first time. This makes a significant contribution to increased utility of the method of protein purification using self-cleavable stimulus responsive tags. </p> <p> When ELP-intein fusion proteins are expressed in bacteria, formation of non-native cytoplasmic aggregates (inclusion bodies) is a common problem which affects the yield of target protein. Inclusion bodies are generally assumed to contain misfolded or partially folded protein through exposure of hydrophobic patches and the consequent intermolecular interactions. Despite a loss of total expression yield and the need for more time, culturing at a lower temperature was reported to promote the expression of genes into soluble proteins and alleviate IB formation. Directly motivated by previous reports, we have applied a low-temperature expression strategy to solve the problems in this research. As expected, most of the T4-ELP inclusion bodies disappeared, and were transformed into a soluble expression, when culturing at lower temperatures. </p> <p> Inverse transition cycling (lTC), as the core method for the system we investigated has proved successful in the past with proteins that were expressed to high levels. However poor level ELP-intein tagged protein expression happens from time to time. It is hypothesized that if an ELP tagged molecule is present in a solution at a very low concentration, adding an excess amount of free ELP to the sample would form hybrid aggregates via the interaction of ELP moieties of the two molecules. We used this efficient and reversible capture system for low yield recombinant protein purification, and found it is perform very well. </p> / Thesis / Master of Applied Science (MASc)

Elastin-like polypeptides (ELPs) consist of simple pentapeptide repeats which can be easily modified by substituting various amino acid residues to control its properties. This provides an ideal platform for studying hydrophobic collapse and secondary/tertiary structure formation. In this thesis, the collapse process of ELP was studied with differential scanning calorimetry (DSC). In DSC thermal cycling, a clear conformational transition was observed. Also, a transiently stable state of ELP V5-120 was noted and it was found that the formation of this state was related to temperature, ramping rate and stabilization time. To explain this, a conformational redistribution model is proposed in which there are two conformations in the ELP solution below its transition temperature. However, after the system is heated up and cooled back down, one of the conformations remains the same while the other one changes to two new conformations. After the conformational distribution is done, the ELP stays in a transiently stable state before gradually shifting back to the original, pre-heat-treatment state. Bi-Gaussian fitting was used to fit DSC response curve and monitor the changes of the different conformations in the system. The influence of ramping rate on the process of conformational redistribution was explained through the equilibration time at each temperature point through heating and cooling. Overall, the ELP V5-120 system is in a dynamic conformational equilibrium, and the equilibration time is much longer than earlier expectations.

The specific manner in which ions associate with a peptide surface is a subject of much research. The models currently proposed tend to rely either on computational results from overly simplified systems, or on observations of bulk solution behavior not applicable to peptide solvation. Herein we demonstrate a new platform for directly measuring specific ion interactions with peptides and use a pair of highly conserved model peptides to investigate specific mechanisms by which ions interact with a peptide surface. A system for investigation of charge selective ion-peptide interactions using a conical glass nanopore was designed. The glass nanopore was coated using layer-by-layer depositions of poly(diallyldimethylammonium) chloride and sodium poly(styrenesulfonate) to control the size and charge selectivity of the nanopore. The tip of this nanopore probe was encapsulated in a 5% agarose gel to prevent peptide fouling. This probe was then used to measure the partitioning of cations to or from the surface of two model peptides: nonpolar V5-120 and positively charged KV6-112 elastin-like polypeptide (ELP). Partitioning was measured by clamping the current through the pore at zero amps and measuring the resulting potential across the nanopore. This potential was used to determine the bulk concentration of electrolyte in a 1 mg/mL peptide in 0.1 M electrolyte solution. Measurements were made with a patch clamp using chloride salts with the cations potassium, lithium, cesium, ammonium, and guanidinium at both room temperature and in an ice bath to ensure that the peptides were in their unfolded state and thus that all possible binding sites were exposed to the solution. All salts were observed to partition to the peptide surface with much less affinity than water, resulting in an increase in the bulk electrolyte concentration with the exception of ammonium, which showed a greater affinity than water for the KV6-112 ELP in the ice bath measurements. These results demonstrate that cations do not favorably partition to nonpolar or cationic peptide surfaces.

This dissertation focuses on the study of inverse temperature transition processes of the poly(N-isopropylacrylamide) (PNIPAM) and the elastin-like polypeptides (ELPs). A novel temperature jump microfluidic system is introduced and this system shows the ability to measure the kinetics of the PNIPAM and the ELPs collapse without a heat transfer problem. The conformational change of the ELPs during the phase transition process is utilized as a nanoscale protein filter to modulate ligandreceptor binding events on supported lipid bilayers (SLBs). This research study is divided into three main parts. The first part is the development of the temperature jump microfluidics. The kinetics of PNIPAM collapse is used as a model system to show the capability of this new device to measure millisecond time scale phase transition processes. The effects of salts on the kinetics of PNIPAM collapse are also shown in this part. To our knowledge, this is the first study which shows the effects of salts on PNIPAM collapse kinetics. The second part of this research is the application of the novel temperature jump microfluidics. The hydrophobic collapse of ELPs composed of identical sequence but different chain length is investigated. By controlling the molecular weight of the ELPs, the thermodynamic contributions from intermolecular hydrophobic interactions, and intramolecular hydrophobic interactions could be calculated individually for this unique system. The third part is the application of the phase transition property of ELPs. The ELPs are conjugated on the surface of the SLBs as a nanoscale protein filter. The conformation of the ELPs can be modulated by ionic strength of the buffer solution or ambient temperature. The ELPs conjugated SLBs platform showed the ability to block IgG binding to biotin conjugated on the SLBs when the ELPs were in the extended coil state and open the access for protein to bind to biotin in compact globule conformation.

Effective methods for isolation and purification of glycoproteins are of increasing significance to the rapidly growing biopharmaceutical and diagnostic industry. Glycoproteins represent the majority of therapeutic proteins on the market and are effectively used to treat immune disorders, infections, cancers, and other diseases. Targeting these glycoproteins is also critical to an emerging field of glycoproteomics aimed to understand structure-function relationships of glycans. Architecturally, these glycoproteins are proteins with covalently linked oligosaccharide chains of varying monosaccharide composition. Affinity chromatography has proven to be an excellent method of glycoprotein purification at the bench scale. However, chromatography in large scale production has its drawbacks. Column fowling, flowrate limitations, and diffusional constraints collectively hinder the effectiveness of the method. An alternative proposed in this dissertation is the use of affinity precipitation as a purification technique. The three main objectives are 1) develop and produce dual-functional, thermo-responsive affinity ligands from a biological host, 2) characterize and optimize the accompanying affinity precipitation method, and 3) apply the ligand and process to relevant, unmodified glycoproteins. The design of the thermo-responsive affinity construct was comprised of two main functional domains. The binding capability was achieved by selection of small ligands with affinity to a specific monosaccharide moiety. Two different lectins, or sugar binding proteins, were used in the fusion design: a fucose binding lectin from Ralstonia solanacearum, and a sialic acid binding lectin from Vibrio cholera. The thermo-responsive functionality was obtained by use of an elastin-like peptide (ELP), which confers inverse solubility relationship properties to the fusion construct. A small library of varying ELP chain lengths were designed to find the optimal size fusion for both production and function. These dual functional ligands were cloned and expressed in the microbial host, E. coli. Furthermore, secretion of these constructs was achieved by employing the Tat secretion pathway in combination with an outer membrane lipoprotein deletion mutant with a leaky periplasm phenotype. This secretory mechanism allows for easy isolation, avoidance of inclusion bodies, and no additional protease inhibitors. After successful production, the ligands were tested to confirm that dual functionality was preserved in fusion form. Once binding conditions and precipitation properties were ascertained, the purification ability was tested on model glycoproteins. Experimentation was carried out monitoring the purification yield, purity, and retained activity of the target enzymes. High contaminant solutions, such as cell lysates, were spiked with the model glycoproteins to mimic crude protein solutions. The purification ability of the constructs in these models was observed. The method was then implemented on two relevant glycoprotein applications: 1) purification of soybean peroxidase from a crude protein extract and 2) targeting the therapeutic protein erythropoietin from albumin rich, used CHO cell media. By implementation of the fucose targeting fusion construct, the unmodified soybean peroxidase is isolated from a natural crude extract from the soybean hull, a by-product of the soybean industry. The affinity precipitation method parameters were optimized with respect to ratios, temperatures, recycle, and elution buffers to achieve successful isolation of the low abundance enzyme. Under the optimized conditions, >95% recovery yield and a purification of 22.7 fold of an active, pure product was attainable. The purification of erythropoietin led to additional experimentation with high-abundant glycoprotein solutions, as well as expansion of the affinity ligand platform. The concept of multi-lectin affinity precipitation, using the fucose and sialic acid binding lection sequentially, was introduced and tested for purification capability. An industrially relevant scheme involving isolation of the erythropoietin from used CHO cell media allowed for an achievable yield of about 60%, with a resulting albumin depletion of about 85%. In addition to development of a pair of novel thermo-responsive affinity ligands for glycoprotein purification, this dissertation provides insight on possible improvements and future directions with respect to the thermo-responsive affinity ligand platform. This unique concept employs novel lectin fusions to target valuable glycoproteins using a method avoiding the major drawbacks associated with chromatography.

Over the past few years, researchers have focused their attention on the development of targeted cancer therapies to minimize the side effects associated with non-targeted treatments such as chemotherapy. Specifically, these approaches have focused on blocking growth factor receptors (GFR) that are overexpressed in cancer cells. In this thesis, we also focus on targeting overexpressed GFR; however, instead of blocking the GFR, our novel approach aims at using them to selectively enhance the endocytotic process of macropinocytosis to deliver peptides that either disrupts the mitochondria or inhibits glycolysis. Herein, we show the selective enhancement of macropinocytosis by the fusion protein comprised of the keratinocyte growth factor (KGF) fused to elastin like polypeptide (ELP), KGF-ELP. Furthermore, we report the synthesis of the fusion protein consisting of mitochondriotoxic peptide (KLAKLAK)2 with ELP, (KLAKLAK)2-ELP. We show that (KLAKLAK)2-ELP forms nanoparticles (NPs) that are internalized via macropinocytosis and their internalization is facilitated by the interaction between the ELP domain and heparan sulfate proteoglycan (HSPG) on the cell surface. This internalization results in mitochondrial swelling, depolarization and subsequent cell death. Moreover, we show that heterogeneous NPs comprising of the two fusions KGF-ELP and (KLAKLAK)2-ELP selectively kill lung cancer cells expressing the keratinocyte growth factor receptor (KGFR). We also report the synthesis of the fusion consisting of peptides derived from a phosphorylated domain of the glycolytic enzyme phophoglycerate mutase (PGM) and ELP, PGM-ELP. We demonstrate that this fusion inhibits the step in glycolysis that converts 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG); the results show that cell death occurred preferentially in lung cancer cells compared to normal cells. Additionally, the heterogeneous NPs comprising of KGF-ELP and PGM-ELP selectively enhanced killing in lung cells with high levels KGFR. Finally, the synthesis of a fusion proteins consisting of four PGM domains fused to ELP, (PGM)4-ELP, exhibits higher cytotoxic effect and efficiency when compared to the single PGM domain fusion, PGM-ELP. Overall, we conclude that targeting overexpressed growth factor receptors to stimulate macropinocytosis can be a tremendously selective therapy for the treatment of lung cancer. This can result in attenuating side effects and improvement of the patient’s prognosis

The aim of this study originated with my role as a teacher wanting to research the impact of the European Language Portfolio, ELP, (2001) on a small group of minority language students, in Brussels’ Flemish region. In this exploratory case study, the European Language Portfolio (Spanish model) was administered to a small group of forty-five primary school students from the Bicultural Foyer Project in Brussels. In spite of the students’ positive attitudes towards this portfolio, the results suggest that the ELP has no validity for them because the Brussels’ Flemish educational community does not offer the appropriate conditions for a possible broader use of this portfolio. Belgium’s language policy environment, including its language policies, is the fundamental reason for the non-implementation of the European Language Portfolio.

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<div>Wounds are created in soft and hard tissue through surgery or disease. As the wound heals, the tissue is held in place using sutures or staples for soft tissue or plates, pins, or screws for hard tissues. These fixation methods inherently damage the surrounding healthy tissue. Surgical adhesives are a non-damaging alternative to these methods. In order to be effective, surgical adhesives must be biocompatible,</div><div>adhere strongly in a moist environment, and have mechanical properties similar to those of the native tissue.</div><div><br></div><div><div>To address the design criteria for surgical adhesives, we look to nature to find inspiration from compounds that provide these properties. Mussels use catechol-based</div><div>molecules to adhere to surfaces in wet and turbulent environments. Incorporating catechols into polymer systems can provide adhesion even in moist biological environments.</div><div>Mimics of elastomeric proteins from soft tissue can be used as backbones for soft and flexible adhesive systems. In particular, elastin-inspired proteins have a well-defined modular sequence that allows for a range of design choices. In this work, we explored the behavior of elastin- and mussel-inspired natural and synthetic polymers in biologically relevant environments.</div></div><div><br></div><div><div>First, the cytocompatibility of a catechol-containing poly(lactic acid) (cPLA) hard tissue adhesive was studied. The cPLA polymer was reacted with iron- or periodatebased</div><div>crosslinkers and compared to PLA. Fibroblasts grown directly on cPLA or cultured with leachate from cPLA had high viability but slower growth than cells on PLA. The periodate crosslinker was significantly cytotoxic, and cells grown on cPLA crosslinked with periodate had reduced metabolism and slowed growth. Cells grown on or in leachate from iron-crosslinked cPLA had similar viability, metabolism, and growth to cells on or in leachate from cPLA. The iron-crosslinked cPLA is a promising</div><div>cytocompatible adhesive for hard tissue applications.</div></div><div><br></div><div><div>Second, two elastin-like proteins (ELP) were developed that had pH-sensitive properties in solution and when crosslinked into hydrogels. Both ELPs had a large number of ionizable tyrosine and lysine residues, and one design also had a large number of ionizable histidine and aspartic acid residues. The stiffness of the hydrogels was maximized at pH values near the isoelectric point of the protein. The stoichometric ratio of crosslinker used affected hydrogel stiffness but did not significantly alter the pH-sensitivity of the gel. The crosslinked gel shrank when swelled at physiological pH. The pH-sensitive mechanical properties of hydrogels made from the two ELPs did not vary significantly. The tyrosine and lysine residues in one ELP were also</div><div>chemically blocked through acetylation to lower the isolectric point of the protein. The acetylated hydrogels had maximum stiffness at a pH near the isoelectric point of the acetylated ELP. The stiffness of both the native and acetylated gels were within the range of soft tissue. Through a combination of crosslinker ratio and chemical modification, the pH-responsive properties of the elastin-inspired hydrogels could be tuned.</div></div><div><br></div><div><div>Finally, adhesive proteins were created that were inspired by both elastin and mussels. An ELP was modified to include catechol groups (mELP). The ELP and mELP were optimized for adhesive use in a soft tissue system. A warm and humid environment was used to study the adhesion of these proteins on pig skin. Iron and (hydroxymethyl) phosphine crosslinkers increased the adhesive strength of both proteins, and periodate increased the adhesive strength of mELP. The adhesive strengths of the proteins were maximized when mELP was mixed with iron or when either protein were mixed with (hydroxymethyl)phosphine crosslinkers. These maximized adhesives were 12-17 times stronger than a commercially available sealant. In addition,</div><div>the iron and mELP adhesive formulation achieved high adhesive strengths even when cured for only ten minutes. This adhesive formula shows promise for adhesive</div><div>applications on soft tissue.</div></div>