Summary
We have previously reported the expression of both full-length recombinant lactoferrin and the recombinant N-lobe half-molecule in baby hamster kidney (BHK) cells (Stowell et al, 1991; Day et al., 1992). The properties of the full-length recombinant protein produced in this system were virtually indistinguishable from those of the native protein isolated from human milk, except for an increased resistance of a minor fraction of the protein to deglycosylation by PNGase. The N-lobe recombinant protein has been characterized (Day et al., 1992) and the structure determined by X-ray crystallography (Day et al., 1993).
These studies have shown the utility of the BHK cell system as a means of producing recombinant lactoferrins. We have now extended our work on recombinant human lactoferrin by the construction and expression of cDNAs for a number of mutant forms of the protein, both in the full-length molecule as well as in the N-lobe half-molecule. These mutants fall into a number of classes and have been designed to address questions related to various aspects of the structure and function of the protein. Some of these mutant classes are summarized below.
Iron binding ligand mutations have mostly been introduced into the N-lobe half-molecule with the ultimate objective of producing an N-lobe protein that is no longer capable of binding iron. Our strategy has largely been to introduce the corresponding residues found in the nonfunctional C-lobe of melanotransferrin (Baker et al, 1992). However, a number of other iron ligand mutants have been made.
For nonglycosylated lactoferrins, the single site for N-glycosylation in the N-lobe half-molecule has been changed in two mutants (LfN:N137A and N137S). Two of the three potential sites in the full-length molecule have been mutated (hLf:N137A; N478A). These residues have been suggested (Spik et al., 1985) as the two sites for glycosylation in the native protein. The cDNA for expression of the triple mutant (hLf:N137A; N478A; N623A) has also been prepared.
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Tweedie, J.W., Baker, E.N., Day, C.L., Sheth, B., Nicholson, H.H. (1997). Mutagenesis of Human Lactoferrin and Expression in Baby Hamster Kidney Cells. In: Hutchens, T.W., Lönnerdal, B. (eds) Lactoferrin. Experimental Biology and Medicine, vol 28. Humana Press. https://doi.org/10.1007/978-1-4612-3956-7_6
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