Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth
- 242 Downloads
Today there is a concern about the use of animal source proteins and peptides in cell culture applications due to potential contamination by adventitious infectious pathogens. Recombinant production of these proteins using a plant host provides a safe and cost effective alternative. In this paper, we tested the effect of rice-derived recombinant human lactoferrin (rhLF) on mammalian cell growth. The purified rhLF was partially (about 50%) iron-saturated (pis-rhLF). Chemical modification of pis-rhLF generated apo-rhLF (<10% iron saturation) or holo-rhLF (>90% iron saturation). All three forms of rhLF (pis, apo, holo) promoted growth of intestinal cells (HT-29) measured as [3H]-thymidine incorporation or viable cell count, but holo-rhLF was most effective. Holo-rhLF was further tested on hybridoma, osteoblast, and human embryonic kidney cells. Results showed that holo-rhLF promoted cell growth and reduced cell doubling time. The concentration of holo-rhLF in media was critical in promoting cell growth and each cell line had different concentration dependence with the most effective range from 5 to 200 mg/L. The effect of rhLF on antibody production was determined using a hybridoma cell line. Significantly, more antibodies were produced by cells grown with holo-rhLF than cells grown without holo-rhLF. We also compared the effect of holo-rhLF to that of human transferrin, a component commonly used in cell culture media as an iron source. Holo-rhLF was as effective as human transferrin in promoting cell growth and antibody production. Considering all the data obtained, we conclude that rhLF from rice is effective in promoting mammalian cell growth and increasing cell productivity.
KeywordsGrowth factor Growth enhancement Serum-free media Animal-free protein Apoptosis
This work was partially supported by a grant from NIH 1 R43 AG026206-01.
- Azuma N.; Mori H.; Kaminogaea S.; Yamauchi K. Stimulatory effect of human lactoferrin on DNA synthesis in BALB/c 3T3 cells. Agric Biol Chem 53: 31–35; 1989.Google Scholar
- Cornish J.; Callon K. E.; Naot D.; Palmano K. P.; Banovic T.; Bava U.; Watson M.; Lin J. M.; Tong P. C.; Chen Q.; Chan V. A.; Reid H. E.; Fazzalari N.; Baker H. M.; Baker E. N.; Haggarty N. W.; Grey A. B.; Reid I. R. Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145: 4366–4374; 2004. doi: 10.1210/en.2003-1307.PubMedCrossRefGoogle Scholar
- Grey A.; Banovic T.; Zhu Q.; Watson M.; Callon K.; Palmano K.; Ross J.; Naot D.; Reid I. R.; Cornish J. The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells. Mol Endocrinol 18: 2268–2278; 2004. doi: 10.1210/me.2003-0456.PubMedCrossRefGoogle Scholar
- Kovar J.; Franek F. Hybridoma cultivation in defined serum-free media: growth-supporting substances. I. Transferrin. Folia Biol (Praha) 31: 167–175; 1985.Google Scholar
- Playford R. J.; Belo A.; Poulsom R.; Fitzgerald A. J.; Harris K.; Pawluczyk I.; Ryon J.; Darby T.; Nilsen-Hamilton M.; Ghosh S.; Marchbank T. Effects of mouse and human lipocalin homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair. Gastroenterology 131: 809–817; 2006. doi: 10.1053/j.gastro.2006.05.051.PubMedCrossRefGoogle Scholar
- Testa U. Proteins of iron metabolism. CRC, New York; 2002.Google Scholar
- Yamada K.; Ikeda I.; Sugahara T.; Hashizume S.; Shirahata S.; Murakami H. Stimulation of proliferation and immunoglobulin M production by lactoferrin in human–human and mouse–mouse hybridomas cultures in serum-free conditions. Cytotechnology 3: 123–131; 1990. doi: 10.1007/BF00143674.PubMedCrossRefGoogle Scholar