Characterization of recombinant human lactoferrin secreted in milk of transgenic mice.
Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine alphaS1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis 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 transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation. 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 will exert similar, if not identical, antibacterial and anti-inflammatory activity in vivo.
Nuijens
J H
JH
Pharming BV, Niels Bohrweg 11-13, The Netherlands. jnuijens@pharming.nl
van Berkel
P H
PH
Geerts
M E
ME
Hartevelt
P P
PP
de Boer
H A
HA
van Veen
H A
HA
Pieper
F R
FR
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000737:chemistry*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin