Expression, purification and characterisation of antimicrobial peptides of human and bovine lactoferrins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
Lactoferrin (Lf). a basic. ~80 kDa iron-binding glycoprotein, is a member of the transferrin family. It is present in the milk and other secretory fluids of many, but not all, mammalian vertebrates. Several biological functions have been ascribed to this protein. These include bactcriostastis, modulation of the inflammatory response, iron nutrition, a role as an anti-oxidant and regulation of myelopoiesis. Full-length human lactoferrin has been expressed in BHK cells, many strains of Aspergillus and with limited success in Saccharomyces cerevisiae. The main aim at the start of this project was to express full-length human lactoferrin (hLF) cDNA in the yeast Kluyveromyces lactis on whey-based media. Yeasts of the genus Kluyveromyces have been used for many years in the food industry and are classified as "Generally Regarded As Safe" (GRAS) organisms. K. lactis has impressive secretion capabilities and can grow on whey-based media (which is abundantly available in New Zealand). Attempts were made to sub-clone full-length hLF cDNA into the K. lactis vector, pEPS1 and the S. cerevisiae vector, pYEXS1 and to express the protein. The establishment of stable insert-carrying constructs of these yeast vectors in E. coli turned out to be an unattainable goal. Direct transformation of the ligation mix into K. lactis produced transformants, which secreted human lactoferrin protein products into the media as assessed by the lactoferrin ELISA assay. The secretion of hLF protein products by recombinant K. lactis continued for few generations, but gradually stopped. There are no known reports on the use of these vectors for the expression of any mammalian proteins in yeast. Lactoferrin has antimicrobial activity against a broad range of Gram-negative and Gram-positive bacteria and against fungi. Originally, the antimicrobial effect of lactoferrin was attributed to its ability to tightly sequester two atoms of iron and hence inhibit microbial growth through nutritional deprivation of iron. Recently, an N-terminal peptide called lactoferricin, isolated from the acid-pepsin hydrolysate of lactoferrin was shown to have greater antimicrobial activity than the intact protein. Currently, the only way to obtain native lactoferricins is to isolate the peptides from the acid pepsin-hydrolysate of lactoferrin, which gives very low yields, or to synthesise them by protein chemical methods, which is very expensive on a large scale. So, heterologous expression of both human and bovine lactoferricins in E. coli was attempted in this study. Synthetic DNA fragments encoding both human and bovine lactoferricins and 3'-end variants of these fragments were sub-cloned into E. coli expression vectors. pPROEXHTa, pET-15b and pGEX-4T1. The constructs were designed to express lactoferricins either as poly-His- or as GST-fusion proteins. In all cases the fusion proteins were expressed as inclusion bodies. The inclusion bodies were urea solubilised and purified on appropriate affinity resins. However, none of the recombinant proteins remained soluble after the urea was removed and therefore could not be further characterised. A similar situation was encountered by other investigators who attempted to express cationic peptides in E. coli. Both lactoferrin and lactoferricin have been shown to bind to the lipid A portion of the bacterial cell wall lipopolysaccharide (LPS), induce the release of LPS and kill the bacteria. In this work, five different E. coli strains were shown to have different susceptibility to native lactoferricin B. Transmission electron microscopy studies of the E. coli strains treated with lactoferricin B revealed considerable differences in their membrane ultrastructure. The most susceptible E. coli strain showed a direct outer membrane dislocation and effect on the cytoplasmic contents. A relatively resistant E. coli strain showed an initial formation of 'membrane blisters'. However, after a long exposure to lactoferricin B, a few cells of this strain showed an outer membrane dislocation and effect on the cytoplasmic contents. The formation of 'membrane blisters' might allow the relatively resistant strain of E. coli to reduce the lethal action of lactoferricin B.