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    Scaling up the production of protein nanofibres

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    KangWONGMasterThesis67024686.pdf (9.967Mb)
    Author
    Wong, Kang Yuon
    Date
    2011
    Permanent Link
    http://hdl.handle.net/10092/6734
    Thesis Discipline
    Chemical Engineering
    Degree Grantor
    University of Canterbury
    Degree Level
    Masters
    Degree Name
    Master of Engineering

    Protein nanofibres, commonly known as amyloid fibrils, are emerging as potential biological nanomaterials in a number of applications. Protein nanofibres are a highly ordered insoluble form of protein, which results when a normally soluble protein aggregates via a self-association process. However, researchers are currently faced with several challenges such as finding a cheap source of proteins that can be obtained without expensive purification and optimizing a scalable method of the manufacturing of protein nanofibres. This thesis has identified crude mixtures of fish lens crystallins as a cheap protein source and has optimized methods for large scale production of protein nanofibres of varying morphologies. Results show that by varying the conditions of fibre formation, individual protein fibres can be used as building blocks to form higher order structures. This ability to control the morphology and form higher ordered structures is a crucial step in bottom up assembly of bionanomaterials and opens possibilities for applications of protein nanofibres.

    The method of formation of protein nanofibres was optimized on a bench scale (1.5 mL Eppendorf tubes) and successfully scaled-up to 1 L volume. For larger scale-up volume (i.e. greater than 10 ml), internal surface area was important for the formation of protein nanofibres. The crude crystallin mixture prepared at 10 mg/mL was heated at 80oC in the presence of 10% v/v TFE at pH 3.8 for 24 hours and stored for an additional of 24 hours at room temperature for storage process. Aggregation and precipitation of proteins were observed as the protein solution was added to the pre-heated TFE. The resulting protein nanofibres were characterised using ThT dye binding, TEM and SEM. The TEM images show a network of long and criss-crossing protein nanofibres with individual fibres of approximately 10 to 20 nm in diameter and 0.5 to 1 μm long. These protein nanofibres were prepared in 1 mL centrifuge tubes and were left on the laboratory bench at room temperature. After 5 months, fresh TEM grids of the sample were prepared and visualized using TEM. Interestingly, TEM images show that a number of individual fibres had self-assembled in an intertwining fashion to form large bundles and higher order structures containing bundles of nanofibres up to 200 nm thick.

    Subjects
    amyloid fibrils
     
    protein nanofibres
     
    scale up
     
    crystallin proteins
     
    bionanomaterials
    Collections
    • Engineering: Theses and Dissertations [2264]
    Rights
    https://canterbury.libguides.com/rights/theses

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