Rhodococcus equi is an important pathogen in foals between one to six months of age and is a major cause of death in in these animals. In addition, R. equi has recently emerged as a significant opportunistic pathogen in immunosuppressed humans, especially those infected with HIV. Despite the ability of the organism to survive stressful growth conditions, for example, exposure to elevated temperature and oxygen radicals, the role of heat shock proteins in the pathogenesis of R. equi has not been well documented. In this project we developed and optimised methods to purify the heat shock protein DnaK from R. equi, using a combination of ion-exchange and affinity chromatography. The effectiveness of the purification protocols were assessed using SDS-PAGE and Western-blotting with anti-DnaK antibodies, and the enzymic activity of the purified DnaK was verified with an ATPase assay. ATPase assays were also used to investigate the roles of other heat shock proteins in enhancing the activity of DnaK.

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The aim of the project was to identify positions and amino acids that contribute to improved structure and stability of bispecific ADAPT proteins. During the 20 weeks project period, different amino acid substitutions were analysed to evaluate the effect on the three-helical structure and stability of bispecific ADAPTs targeting human serum albumin (HSA) and tumor necrosis factor α (TNFα). Furthermore, the study also included identification of which amino acid substitutions that affect the simultaneous binding ability of the anti-TNFα ADAPT. The amino acids substitutions that demonstrated improved stability was further evaluated in two other bispecific ADAPT proteins targeting epithelial cell adhesion molecule (EpCAM), in terms of structure and stability. The TNFα-targeting ADAPT variants was produced in Escherichia coli (E. coli), purified through affinity chromatography using a HSA-coupled matrix and was further analysed and evaluated using SDS-PAGE, circular dichrosim, size-exclusion chromatography and surface plasmon resonance to detect expression levels, yields, thermal stability, secondary structure, and simultaneous binding to TNFα and HSA. Furthermore, the production, purification and evaluation were redone with other bispecific ADAPTs targeting EpCAM, to be able to draw more general conclusions. The outcome showed which amino acids substitutions in the scaffold that improve the structure and stability of the TNFα- and EpCAM-binding ADAPT protein variants, respectively. Some of the ADAPT variants targeting TNFα showed improved stability and increased melting temperature. One of the variants with most potential from these mutants was ADAPT_TNFα5_F21K, both able to refold after heat treatment and demonstrated a higher melting temperature in the same order as the original binder. The variant bound HSA but not TNFα, thus consequently was not able to bind TNFα and HSA simultaneously. The variants ADAPT_TNFα5_V17I and ADAPT_TNFα5_M22Q both demonstrated a clear alpha-helix structure, were able to refold after heat treatment and demonstrated simultaneous binding to TNFα and HSA. The melting temperature for ADAPT_TNFα5_V17I was the same as for the original binder (59°C) and ADAPT_TNFα5_M22Q showed a decreased melting temperature (45°C) compared to the original binder. The amino acid substitutions that improve the stability of the original binder was combined and two variants withthese mutations were designed. Unfortunately, these variants could not express in E. coli cells and were not able to be produced. For the EpCAM targeting mutants one variant, ADAPT_EpCAM_02_X11N, showed huge improvements of the stability and structure compared to the original binder ADAPT_EpCAM_02. This variant improved the melting temperature with 24°C compared to the original binder and was able to refold after heat treatment, which the original binder did not have the ability to do. However, ADAPT_EpCAM_02_X11N was not able to simultaneously bind EpCAM and HSA, demonstrating that the mutation also had an effect on the binding ability. In the variant ADAPT_EpCAM_08 the mutation Y5I improved the melting temperature with 14°C compared to the original binder and was able to refold after thermal denaturation. However, the simultaneous binding to EpCAM and HSA was negatively affected. The project results have contributed to better understanding of the bispecific ADAPT proteins, which enables further development of the scaffold. The amino acid positions in the scaffold that showed to be important for ADAPT structure and stability will be used in the design of a new ADAPT-library, from which new binders with improved structure and stability hopefully can be selected, which might have the potentially to be used as future therapeutics. / Syftet med projektet var att identifiera positioner och aminosyror som bidrar till ökad struktur och stabilitet hos bispecifika ADAPT proteiner. Under projektperioden på 20 veckor har olika aminosyrasubstitutioner i ett bispecifikt TNFα/HSA-bindande ADAPT protein analyserats med syftet att undersöka om dessa substitutioner förbättrar stabiliteten och strukturen hos proteinet. Vidare analyserades hur dessa aminosyrasubstitutioner påverkar ADAPTs förmåga att binda HSA och TNFα samtidigt. De aminosyrasubstitutioner som visade på förbättrad stabilitet, utvärderades vidare med avseende på struktur, stabilitet och bindning i två andra bispecifika ADAPTs riktade mot EpCAM. De TNFα-bindande ADAPT-varianterna producerades i E. coli, renades fram med affinitetskromatografi med en HSA-kopplad matris, analyserades och utvärderades med metoder som SDS-PAGE, cirkulär dichroism, gelfiltrering och surface plasmon resonance för att detektera uttrycks-nivåer, utbyten, termisk stabilitet, sekundärstruktur och om proteinerna upprätthöll bindning till både HSA och TNFα. Vidare utvärderas de aminosyrasubstitutionerna som visade på förbättrad stabilitet även i andra bispecifika ADAPTs riktade mot EpCAM för att kunna dra mer generella slutsatser. Resultaten visade vilka aminosyrasubstitutioner i proteinet som förbättrade strukturen och stabiliteten hos de TNFa- respektive EpCAM-bindande ADAPT-protein varianterna. Några av ADAPT-TNFa varianterna visade förbättrad stabilitet och ökad smälttemperatur. ADAPT_TNFα5_F21K hade en tydlig alpha-helix struktur och kunde återveckas efter värmebehandling. Varianten hade en smälttemperatur i samma storleksordning som den ursprungliga TNFα-bindande ADAPT varianten. ADAPT_TNFα_F21K hade dock inte förmågan att binda simultant till TNFα och HSA. Varianterna ADAPT_TNFα_V17I och ADAPT_TNFα_M22Q visade en tydlig alpha-helix struktur, kunde återveckas efter värmebehandling och visade simultan bindning till TNFα och HSA. ADAPT_TNFα_V17I hade samma smälttemperatur som den ursprungliga bindaren (59°C) medan ADAPT_TNFα_M22Q visade en minskad smälttemperaturen (45°C) jämfört med original-bindaren. Två varianter bestående av kombinationer av de aminosyrasubstitutioner som visade på förbättrad stabilitet skapades. Dessa kunde tyvärr inte uttryckas och produceras i E.coli-celler. Gällande de EpCAM-bindande ADAPT-proteinerna var det en variant, ADAPT_EpCAM_02_X11N, som visade stor förbättring i stabilitet och struktur jämfört med originalbindaren ADAPT_EpCAM_02. Denna variant hade 24°C högre smälttemperatur jämfört med originalbindaren och kunde återveckas efter värmebehandling, vilket inte original-bindaren kunde. Dock kunde inte ADAPT_EpCAM_02_X11N binda simultant till EpCAM och HSA. Detta tyder på att denna mutation har en negativ effekt på ADAPTs bindingskapacitet. Varianten ADAPT_EpCAM_08_Y5I visade en förbättrad smälttemperatur på 14°C jämfört med original-bindaren ADAPT_EpCAM_08 och kunde återveckas efter värmebehandling. Dock resulterade även denna mutation i en negativ effekt på den simultana bindningen till EpCAM ochHSA. Projektets resultat har bidragit till en bättre förståelse av de bispecifika ADAPT-proteinerna och möjliggör en vidareutveckling av scaffoldet. Aminosyranspositionerna som visade sig vara viktiga för ADAPTs struktur och stabilitet kommer användas för design av ett ny ADAPT-bibliotek, från vilket nya bindare med förbättrad struktur och stabilitet förhoppningsvis kan bli selekterade. Dessa nya, förbättrade bindare, ökar de bispecifika ADAPT proteiners användningsmöjligheter inom terapi.

With an expanding life-science sector and growing production of recombinant proteins, the need for efficient downstream processing is increasing. Certain proteins are sensitive to the harsh conditions often used in protein purification, such as low pH, which can result in aggregation and denaturation. ZCa is a domain derived from Protein A that can be used for calcium-dependent purification of antibodies without the need for acidic pH. Based on this domain, the CaRA library has been constructed, which targets other therapeutic proteins than human antibodies. Four of the proteins isolated from the CaRA library, namely CaRA_scFv_1, CaRA_scFv_2, CaRA_G-CSF_1 and CaRA_G-CSF_3, are presented here for the purification of single chain variable fragment and granulate colony stimulating factor. The four proteins were produced as monomers, trimers and hexamers in an attempt to increase the binding capacity and attached to a matrix for purification using site-specific coupling. The successful binders CaRA_scFv_1 and CaRA_scFv_2 showed high affinity for their target protein scFv and were able to selectively capture an increased number of molecules through multimerization. Calcium-dependent binding was demonstrated by elution at neutral pH using the calcium chelator citrate, thus concluding that these multimerized CaRA variants can be used to considerably increase the efficiency in scFv purification while providing excellent purity and significantly reducing the risk of aggregation.

<p>DNA integrity is continuously compromised by cellular metabolic activity and environmental factors resulting in many lesions per cell per day (Lindahl et al.,2009). If left unrepaired or if repaired improperly, these lesions pose an obstacle to cellular transcription and replication. In due course, DNA damage may lead to mutations and genomic instability that will eventually threaten the viability of the cell and organism and can lead to tumour formation and neurological disorders (Rass et al., 2006). Aprataxin (Aptx) is a conserved factor in DNA repair required for the repair of damaged DNA following abortive DNA ligation (Date et al., 2001; Moreira et al 2001). Aptx initiates repair by processing adenylated DNA ligation events via its unique AMP lysine hydrolase activity (Rass et al 2006). Inability of Aptx to carry out these functions has been shown to be the causative agent in the neurological diseases, ataxia with oculomotor apraxia (Takahashi et al 2007, Yoon et al 2008, and Ferrarini et al., 2007). Nevertheless, the exact mechanism of Aptx has not been established and the full extent of its function(s) (i.e. binding to DNA or other proteins) is not fully understood. In order to achieve a comprehensive understanding of the mechanism governing Aptx, further functional and structural analysis is required. Here, it was found that Aptx has deadenylation activity that is further supported by the crystal structure of ATPX-DNA-AMP-Zn²⁺. Importantly, work reported in this thesis describes for the first time methods for expression and purification of large quantities of high quality human Aptx from bacterial cells. This protein is shown to possess robust deadenylation activity, suitable for further <em>in vitro</em> screening of small molecule inhibitors.</p> / Master of Science (MSc)

The biopharmaceutical industry has experienced remarkable progress in the upstream production capacity of life-saving proteins. This is while the downstream processing has failed to keep pace, including unit operations which are working close to their physical limit with no economy of scale. Column chromatography which is an integral unit in different stages of downstream purification is considered as the major bottleneck in this section. The packed-bed resin media is costly and the processes are labor-intensive and extremely time consuming. Membrane chromatography which uses a stack of adsorptive membranes as the chromatographic media is one of the most promising alternatives for conventional chromatography techniques. The performance of membrane adsorbers is consistent over a wide range of flow rates which is owing to the dominance of convective solute transport as opposed to the diffusion-based nature of mass transfer within the pores of the resin beads. This translates to much higher productivity and considerably lower buffer consumption (even as high as 95%), leading to much lower overall processing costs. The other advantages are significantly lower footprints and decreased pressure drops, both contributing to diminished capital costs. Membrane adsorbers are greatly scalable and used in a single-use manner. The latter eliminates the cleaning and validation steps and brings about much shorter processing times and higher flexibility in process development. Due to the performance advantages of membrane chromatography, this technique is now widely used in purification of high volumes of samples in late-stage polishing. Currently available membrane adsorbers have radial-flow spiral-wound configuration with high frontal surface area to bed height ratio according to which dilute impurities are removed in a flow-through format at very high flow rates and low pressure drops. Nevertheless, they fail to give high-resolution for bind-and-elute separations which makes them unsuitable for many unit operations, highly restricting their application. Severe design deficiencies such as large dead volumes and varying membrane area over the bed height result in broad and poorly resolved peaks. Herein, a novel device design was successfully developed which addresses the abovementioned shortcomings. The laterally-fed membrane chromatography (LFMC) devices house a stack of rectangular membrane sheets with two rectangular lateral channels on both sides of the stack as the feed and permeate channels. The design offers balanced pressure over the sides of the stack as well as even solute flow path lengths due to which the solute residence time is very uniform. Also, the small dead volumes minimize the dispersion effects. These features make the LFMC technology highly suitable for bind-and-elute applications, the improvement which is brought about by a simple design. The devices are easy to fabricate and highly scalable. The LFMC devices containing cation-exchange (CEX) membranes with 7 mL bed volume were examined for bind-and-elute separation where they outperformed the equivalent commercially available radial-flow devices. The design was further modified to give even lower dead volumes and more cost-effective fabrication. The latest embodiment of the device gave resolutions which were comparable with the ones obtained with the commercially packed resin columns in 1 mL and 5 mL scale with consistency over wide range of flow rates. The results were all acquired using a three component model protein system. Upon the approval of suitability of the device for bind-and-elute separation, the CEX-LFMC was used for purification of monoclonal antibodies (mAbs), the largest class of biopharmaceuticals. The device showed great performance in separation of mAb charge variants when extensively shallow gradients (60 membrane bed volumes) were required. The devices offered very stable conductivity gradients at high flow rates. LFMC devices in three different preparative scales gave great performance in separation of mAb aggregates which was approved for different mAb samples. The other application studied with the CEX-LFMC devices was the single-step preparative purification of mono-PEGylated proteins which is as well very challenging due to the physicochemical similarities between the target molecules and the impurities. Collectively, the LFMC devices combine the high-resolution with high-productivity which is highly desirable in downstream purification of biological molecules with great potential to expand the application of membrane chromatography. Finally, the LFMC devices were modified to adapt the analytical scale where they were integrated with a stack of hydrophilized PVDF membranes. The device successfully delivered ultra-fast separation of mAb aggregates in less than 1.5 minutes based on hydrophobic interaction membrane chromatography (HIMC). The assay times achieved with the HI-LFMC technique outclassed the currently available ultra-high performance chromatography (UPLC) methods at the same time with being extremely cost-effective. The application of the LFMC technology in analytical scale has great potential to offer cheap and rapid analysis in process development and quality control section of biopharmaceutical manufacturing. / Thesis / Doctor of Philosophy (PhD)

Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12. / Ph. D.