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dc.contributor.advisorWall, Gerard
dc.contributor.authorTilbury, Maura A.
dc.description.abstractNaturally-occurring peptides and proteins with diverse biological roles have enormous potential for use as therapeutics and biomaterials. While proteins of biomedical interest were traditionally isolated from their native sources, advances in recombinant protein expression have now enabled safe, efficient and large-scale production of highly pure protein. This project utilised Escherichia coli as an expression host for two recombinant proteins with potential biomedical application: (i) a barnacle cement protein for investigation as a biomimetic glue, and (ii) human superoxide dismutase enzyme for targeted pulmonary delivery. Proteins involved in wet adhesion of marine organisms are of growing biomedical interest due to their potential use as surgical adhesives. Recombinant protein technology allows such adhesive proteins to be produced at the scale needed to investigate their adhesive mechanisms. The 19 kDa cement protein (cp19k) of the stalked barnacle Pollicipes pollicipes was expressed in E. coli BL21. Co-overproduction of E. coli molecular chaperones GroEL-GroES and trigger factor (TF) resulted in increased yields of soluble cp19k protein. Surface coat analysis revealed high adsorption of a cp19k-TF complex on both hydrophobic and hydrophilic surfaces but low adsorption of the recombinant cp19k (rPpolcp19k) protein. The purified recombinant protein also exhibited negligible adsorption on hydrophobic, neutral hydrophilic or charged self-assembled monolayers in surface plasmon resonance assays designed to mimic the barnacle cement gland and seawater conditions. Due to its low adhesive capability, the potential cohesive ability of the protein was investigated, using an amyloid-specific fluorometric assay, revealing self-assembly of the protein into amyloid fibrils under cement gland-like but not seawater conditions. Transmission electron microscopy and atomic force microscopy confirmed and further characterised the rPpolcp19k fibrils. Our findings that rPpolcp19k self-assembles into amyloid fibrils suggest a cohesive role for the protein which could be exploited in biomedical adhesion. Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory failure syndrome that is characterised by increased permeability of the alveolar-capillary membrane, pulmonary oedema and the acute onset of hypoxemia. In healthy individuals, an oxidant-antioxidant equilibrium is maintained by antioxidants such as superoxide dismutase enzymes. During the acute phase of ARDS, however, neutrophil infiltration into the alveolar space results in x uncontrolled release of reactive oxygen species and proteases, overwhelming the antioxidant defences and leading to alveolar epithelial and lung endothelial injury. Several fusion proteins incorporating human Cu-Zn-superoxide dismutase protein (hSOD1) were designed for aerosol delivery by nebulisation. Expression of the hSOD1 fusion proteins in E. coli BL21 was optimised using co-overproduction of GroEL-GroES chaperones. The fusion proteins exhibited high superoxide dismutase activity in fused and unfused formats and protected human bronchial epithelial cells from oxidative damage. The hSOD1 protein retained its activity post-nebulisation and demonstrated a satisfactory lung deposition profile for delivery to the lower respiratory tract. The recombinant hSOD1 exhibited no adverse effects in an in vivo rat model. The results provide a strong basis for further investigation of the therapeutic potential of non-fused hSOD1 protein in the treatment of ARDS.en_IE
dc.publisherNUI Galway
dc.subjectEscherichia colien_IE
dc.subjectrecombinant proteinen_IE
dc.subjectfunctional amyloiden_IE
dc.subjectfusion proteinen_IE
dc.subjectsuperoxide dismutaseen_IE
dc.subjectNatural Sciencesen_IE
dc.titleThe expression of recombinant proteins with biomedical importance in Escherichia colien_IE
dc.contributor.funderScience Foundation Irelanden_IE
dc.contributor.funderEuropean Regional Development Funden_IE
dc.local.noteTwo recombinant proteins were expressed in Escherichia coli: a barnacle cement protein (cp19k) with potential as a biomimetic adhesive and an antioxidant superoxide dismutase (hSod1) fusion protein for the treatment of acute respiratory distress syndrome (ARDS). This work demonstrates the versatility of E. coli for the production of protein therapeutics.en_IE
dcterms.projectinfo:eu-repo/grantAgreement/SFI/SFI Research Centres/13/RC/2073/IE/C�RAM - Centre for Research in Medical Devices/en_IE

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