Characterization of Recombinant Human Lactoferrin Secreted in Milk of Transgenic Mice

Abstract

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 ␣S 1 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.

Monosialylated biantennary N-glycoforms containing GalNAc–GlcNAc antennae predominate when human EPO is expressed in goat milk

“…Also, the charged N-glycans were mainly a2,6-monosialylated with Neu5Ac or Neu5Gc present in a ratio of 1:1 corresponding to a significant increase in terminal Neu5Gc. Only a low amount of disialylated species (Hex 3 HexNAc 6 (NeuAc) 2 and Hex 3 HexNAc 6 dHex(NeuAc) 2 ) were detected. Mostly, N-glycans were core-fucosylated, without evidence of outer-arm fucosylation.…”

“…The compounds found were similar to those identified in the neutral fraction but were mostly monosialylated with Neu5Ac or Neu5Gc. Evidence of disialylation was observed for N-glycans with compositions Hex 3 HexNAc 6 (NeuAc) 2 (m/z 1211.7), and Hex 3 HexNAc 6 dHex(NeuAc) 2 (m/z 1284.7).…”

“…To date several proteins have been expressed in the milk of transgenic animals in quantities ranging from a few micrograms to several grams per liter. Some examples are: human lactoferrin in mice [2] and cows [3]; a1-antitrypsin [4] and C1 inhibitor [5] in rabbits; human anti-thrombin [6] and human tissue-type plasminogen activator [7,8] in goats; human Factor VIII [9] and human Protein C [10] in pigs; human a-glucosidase in mice [11] and rabbits [12] and human erythropoietin in rabbits [13] and pigs [14]. However, the functionality and specificity of mammary gland glycosylation is, as yet, incompletely understood in comparison with other mammalian cell lines.…”

Enhanced Th1 Response toStaphylococcus aureusInfection in Human Lactoferrin-Transgenic Mice

“…hLf-transgenic mice (line 2261) and wild-type littermates (strain B6CBA) generated as previously described (16,17) were used to establish a colony at Glasgow University (Glasgow, U.K.). For the CIA model, the transgenic mice were backcrossed on to the DBA/1 background and used after six backcrosses.…”

Allergenicity Assessment of a Genetically Modified Protein-Recombinant Human Lactoferrin

“…However, the yields have been low and since hLF is a highly folded, cross-linked and glycosylated protein, there has not been good success. A recent publication demonstrated successful production of rhLF in transgenic mice with production targeted to the mammary gland [30,31]. A previous attempt to produce rhLF in rice has so far not been fully successful as the plant glycosylation system modifies added asparagine linked glycans differently than mammalian cells and regulators of GMO's (Genetically Modified Organism's) have not approved the GM (Genetically Modified) rice as acceptable.…”