Expression and characterisation of the n-terminal half of human lactoferrin : [t]his thesis is submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry

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Lactoferrin is an 80 kDa iron binding protein which is found in human milk and other exocrine solutions. Each molecule contains two metal binding sites which each bind a single iron atom with high affinity. The properties of the two sites are slightly different. In an attempt to more fully understand the nature of these differences a construct for the high level expression of the N-terminal half of human lactoferrin (LfN) has been constructed and protein expressed from this construct has been purified and characterised. Characterisation of the recombinant protein has shown that the signal peptide is correctly removed from LfN and that an N-linked carbohydrate moiety is added to LfN. LfN has been shown to bind one iron atom and the spectral properties are very similar to those of Fe2Lf. The most significant difference between hLf and LfN is in the pH stability of iron binding. Iron is released from LfN 2 pH units higher than from hLf. In an attempt to understand the bases for this difference a structural analysis of LfN was initiated. Using deglycosylated protein high quality crystals of both iron free and iron saturated LfN have been grown. The structures of both FeLfN and ApoLfN have both been solved by molecular replacement using the coordinates from the N-lobe of Fe2Lf as the starting model. The structure of FeLfN has been refined using data between 8.0 and 2.0 Å. The current model has good geometry and is believed to accurately represent the structure of FeLfN. The structure of FeLfN provides the highest resolution and most accurate structure of a member of the transferrin family. Analysis of the structure has shown that the folding pattern and the environment of the iron atom in FeLfN are very similar to the N-lobe of Fe2Lf although several differences exist. Most of the differences seen are due to the absence of the C-lobe and the rearrangement of residues 315 - 327. The altered conformation of residues 315 - 327 and the changes in the solvent accessibility to other residues are believed to be responsible for the different iron binding and release properties of LfN. Although the structure of ApoLfN is not complete analysis of this structure has shown that unlike the N-lobe of intact apo hLf the domains are closed in ApoLfN. The structure of ApoLfN is very similar to that of FeLfN even though the crystal packing is quite different. In addition although the protein was believed to be iron free there is some density in the iron site which is unaccounted for at present. This study continues. Several mutants of LfN have also been created. These mutants have shown that the carbohydrate groups attached to lactoferrin probably have a role in folding and secretion of lactoferrin by BHK cells. Several mutants involving changes to residues involved in metal and anion binding have also been created. These mutants have helped us begin to define the changes responsible for preventing iron binding in the C-lobe of melanotransferrin. In addition the role of arginine 121 has been investigated however further analysis of these mutants is required before the structural changes responsible for the different properties can be defined.