CHARACTERIZATION OF RECOMBINANT HUMAN LACTOFERRIN SECRETED IN MILK OFTRANSGENIC MICE

Human lactoferrin (hLF) is an iron-binding protein involved in host de fense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to r egulatory elements of the bovine alpha S-1 casein gene. Recombinant hL F expressed in the milk of transgenic mice (transgenic hLF) was compar ed with natural (human milk-derived) hLF. Immunological identity of th e two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunizati on with natural hLF. Mono S cation-exchange chromatography and N-termi nal protein sequencing of transgenic and natural hLF revealed identica l cationicity and N-terminal sequences. SDS-polyacrylamide gel electro phoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF, Unsaturated tr ansgenic hLF could be completely resaturated upon addition of iron. Sl ight differences in mobility between transgenic and natural hLF on SDS -polyacrylamide gel electrophoresis were abolished by enzymatic deglyc osylation, Binding of transgenic and natural hLF to a range of ligands , including bacterial lipopolysaccharide, heparin, single-stranded DNA , Cibacron blue FG 3A, and lectins, was not different. Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF wi ll exert similar, if not identical, antibacterial and anti inflammator y activity in vivo.