Biochemistry of Protein Production Bioprocess
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Professor Paul Dalby
Two-hour written examination (70%)
One 3,000 word essay (30%)
The course will introduce protein biochemistry, with related analytical and molecular biology techniques, specifically tailored for biochemical engineers. Fundamentals of protein expression, purification, structure, folding and stability will underpin case studies in protein manufacturing, including purification, formulation and associated problems such as aggregation.
Techniques for analysing proteins will include “high-end” methods such as crystallography and NMR, but also a range of techniques used in bioprocess monitoring and quality control such as analytical chromatography, ELISA, SDS-PAGE, isoelectric focusing, dynamic light scattering, UV absorbance, and circular dichroism. Basic molecular biology techniques for modifying protein expression constructs will also be introduced.
Introduction to proteins:
Variability in protein properties (size, shape, location in cells)
Protein types (eg structural, transport, carriers, enzymes, signalling, motor proteins, receptors, chaperones etc)
Applications of proteins in biotechnology (Catalysis, Therapy, Molecular Biology)
Amino acids and their properties, primary sequence, polypeptide dihedral angles, Ramachandran plot, secondary structure properties and bonding, super-secondary structure, tertiary structure, domains, motifs, and quaternary structure. Cofactors and post-translational modifications.
Protein properties: molecular weight, charge, pI, hydrophobicity, solubility, precipitation and salt effects.
Protein Analysis: SDS-PAGE, Western blotting, IEF, UV-Visible spectroscopy, Analytical Chromatography, CD, fluorimetry, DLS, AUC, LCMS.
Protein structure determination: EM, NMR and crystallography basics.
Monoclonal antibody structure and therapeutic applications
Protein stability: denaturation, refolding, free-energy of unfolding, measurement of melting temperatures, predicting stability from potential functions, aggregation, oxidation, Deamidation.
Bioprocess monitoring: PAT, critical quality attributes, analytical methods in bioprocessing.
Protein formulation: forced degradation, Arrhenius and non-Arrhenius behaviour, excipient types, freeze-drying.
Enzymes: biocatalysis, enzyme kinetics, inhibition.
Protein expression: vectors, strains, promotors.