Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

A study of water binding, mobility and dehydration in contact lens hydrogels using NMR

Hydrogel polymers are used for the manufacture of soft (or disposable) contact lenses worldwide today, but have a tendency to dehydrate on the eye. In vitro methods that can probe the potential for a given hydrogel polymer to dehydrate in vivo are much sought after. Nuclear magnetic resonance (NMR) has been shown to be effective in characterising water mobility and binding in similar systems (Barbieri, Quaglia et al., 1998, Larsen, Huff et al., 1990, Peschier, Bouwstra et al., 1993), predominantly through measurement of the spin-lattice relaxation time (T1), the spinspin relaxation time (T2) and the water diffusion coefficient (D). The aim of this work was to use NMR to quantify the molecular behaviour of water in a series of commercially available contact lens hydrogels, and relate these measurements to the binding and mobility of the water, and ultimately the potential for the hydrogel to dehydrate. As a preliminary study, in vitro evaporation rates were measured for a set of commercial contact lens hydrogels. Following this, comprehensive measurement of the temperature and water content dependencies of T1, T2 and D was performed for a series of commercial hydrogels that spanned the spectrum of equilibrium water content (EWC) and common compositions of contact lenses that are manufactured today. To quantify material differences, the data were then modelled based on theory that had been used for similar systems in the literature (Walker, Balmer et al., 1989, Hills, Takacs et al., 1989). The differences were related to differences in water binding and mobility. The evaporative results suggested that the EWC of the material was important in determining a material's potential to dehydrate in this way. Similarly, the NMR water self-diffusion coefficient was also found to be largely (if not wholly) determined by the WC. A specific binding model confirmed that the we was the dominant factor in determining the diffusive behaviour, but also suggested that subtle differences existed between the materials used, based on their equilibrium we (EWC). However, an alternative modified free volume model suggested that only the current water content of the material was important in determining the diffusive behaviour, and not the equilibrium water content. It was shown that T2 relaxation was dominated by chemical exchange between water and exchangeable polymer protons for materials that contained exchangeable polymer protons. The data was analysed using a proton exchange model, and the results were again reasonably correlated with EWC. Specifically, it was found that the average water mobility increased with increasing EWe approaching that of free water. The T1 relaxation was also shown to be reasonably well described by the same model. The main conclusion that can be drawn from this work is that the hydrogel EWe is an important parameter, which largely determines the behaviour of water in the gel. Higher EWe results in a hydrogel with water that behaves more like bulk water on average, or is less strongly 'bound' on average, compared with a lower EWe material. Based on the set of materials used, significant differences due to composition (for materials of the same or similar water content) could not be found. Similar studies could be used in the future to highlight hydrogels that deviate significantly from this 'average' behaviour, and may therefore have the least/greatest potential to dehydrate on the eye.