Lactoferrin recombinant

Van Berkel, P.H., Welling, M.M., Geerts, M. et al. (2002) Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nature Biotechnology, 20 (5), 484—487. 

Since that time many more sequences have become available through the advent of recombinant DNA technology and the deduction of amino acid sequences from the base sequences of cloned DNA. At the present time, the primary structures (amino acid sequences) of 14 proteins of the transferrin family have been established. These include seven serum transferrins, from human 10, 36), pig (37), horse 38), rabbit 39), toad Xenopus laevis) 40), sphinx moth (M. sexta) 13), and cockroach Blaberus discoidalis 4) chicken 34, 35) and duck 41) ovotransfer-rins four lactoferrins, from human (11, 42), mouse 43), pig 44) and cattle 45, 46) and the human tumor cell melanotransferrin 47). All of these sequences are available from sequence databases such as EMBL and SWISSPROT. 

Complementing the structural studies of the intact transferrins, a number of fragments have also been crystallized, including proteolytic N-terminal half-molecules of rabbit serum transferrin (69) and chicken ovotransferrin (70), recombinant N-terminal half-molecules of human lactoferrin (71) and human serum transferrin (72), and a quarter-molecule fragment of duck ovotransferrin (73). All of these have now led to high-resolution structures (74-77). 

Coordinates for human lactoferrin, in both diferric (78) and apo- (80) forms, for diferric rabbit serum transferrin (68), and for the three fragment structures, the proteolytic N-lobe of rabbit serum transferrin (74), the recombinant N-lobe of human lactoferrin (75) and the duck ovotransferrin quarter-molecule (76), all in their iron-bound forms, can be obtained from the Brookhaven Protein Data Bank (Brookhaven National Laboratory, Upton, New York). 

The bilobal structure of transferrins means that half-molecules, representing either the N-terminal or C-terminal lobe, can be relatively easily prepared, either by limited proteolysis or by recombinant DNA methods (Section III.A). Relatively high-resolution crystal structures have been determined for three such half-molecules, the proteolytic N-lobes of rabbit transferrin (74) and chicken ovotransferrin (77) at 2.3 A and the recombinant N-lobe of human lactoferrin at 2.0-A resolution (75). These show that both the protein structure and the metal and anion binding sites are the same as in the intact parent structures. In fact comparison of the metal and anion sites of the lactoferrin and transferrin half-molecules with each other and with the N-lobe of lactoferrin shows very close correspondence 92 atoms from the nine residues, plus metal and anion, making up the immediate binding site can be superimposed with an rms deviation of only 0.4 A (75). 

Fig. 28. The pH dependence of iron release from human serum transferrin (Tf), human lactoferrin (Lf), and the recombinant N-terminal half-molecule of human lactoferrin (Lfm). Also shown is a plot (dashed line) for the release of cerium from Ce4+-substituted lactoferrin, demonstrating the increased difference between the two sites for metal ions other than Fe3+.

The recombinant whole molecules are both expressed in glycosylated form, although the glycosylation patterns differ from the proteins isolated from natural sources. The recombinant human transferrin binds to receptors both in its glycosylated form and as a nonglycosylated mutant, showing that the carbohydrate is not required for receptor binding (230). Recombinant human lactoferrin shows identical spectroscopic properties and shows an identical profile of pH-dependent iron release when compared with human milk lactoferrin (231). 

Researchers (Nuijens et al., 1997) at the Leiden University (the Netherlands) in collaboration with Pharming, NV (Leiden, the Netherlands) compared recombinant human lactoferrin (rhLF) expressed in the milk of transgenic mice with natural human milk-derived lactoferrin (hLF). They concluded that the unsaturated rhLF and natural hLF had comparable properties, indicating that hLF produced in bovine milk will exert similar, if not identical, antibacterial and anti-inflammatory activities in vivo. Pharming also developed the first transgenic bull in the late... 

Phase I studies conducted by Pharming indicated that cows milk-derived recombinant human lactoferrin (rhLF) is well tolerated at high doses. Volunteers were injected intravenously with rhLF without negative side effects. An oral biodistribution study in human volunteers has shown that natural and recombinant hLF behave in a similar fashion in the digestive tract. 

Ventria is producing rhLF and recombinant human lysozyme (rhLZ) accumulating in rice seed at a level of 5 g kg flour weight [382]. Rice-derived rhLF and rhLZ were shown to be identical to the human proteins, and to have stability similar to native human lactoferrin and lysozyme when exposed to heat, pH changes, and in vitro digestion [308, 314, 383]. Both lactoferrin and lysozyme are multifunctional... 

Suzuki YA, Kelleher SL, Yalda D, et al. Expression, characterization, and biologic activity of recombinant human lactoferrin in rice. J. Pediatr. Gastroenterol. Nutr., 2003 36(2) 190-199. 

Bethell DR, Huang J. Recombinant human lactoferrin treatment for global health issues iron deficiency and acute diarrhea. Biometals, 2004 17(3) 337-342. 

California-based Ventria Bioscience (Sacramento) is at the stage of field trials of transgenic rice plants expressing recombinant lactoferrin and lysozyme in rice seed (see Part IV, Chapter 8). It is worth mentioning, that these products might reach market later than planned considering difficulties with obtaining the necessary field trial permits [39-41]. 

Meristem Therapeutics (Clermont-Ferrand, France) has a gastric lipase, produced in grains of corn, for the treatment of cystic fibrosis in Phase II clinical trials. The company also has a recombinant lactoferrin at the stage of preclinical trials, in addition to human serum albumin, collagen and monoclonal antibodies at R D stage. 

Zavaleta, N., Figueroa, D., Rivera,)., Sanchez,)., Alfaro, S., and Lonnerdal, B. (2007) Efficacy of rice-based oral rehydration solution containing recombinant human lactoferrin and lysozyme in Peruvian children with acute diarrhea./. Pediatr. Gastroenterol. Nutr., 44, 258-264. 

The use of genetically modified higher plants that produce recombinant products in their leaves, fruits, roots, or other parts is a second option. Transgenic plants are under serious consideration for what is called molecular farming or plant-made pharmaceuticals for products such as insulin, lactoferrin, trypsin, secondary metabolites, and non-pharmaceutical products such as bioplastics. 

Engelmayer, J. Varadhachary, A. Compositions comprising recombinant lactoferrin and its variants in the treatment of diabetes mellitus. PCX Int. Appl. WO 2004103285, 2004 Chem. Abstr. 2004, 141, 420455. 

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