Affiliation
a Institute of Materials Research and Engineering, (IMRE, A*STAR), Innovis, Singapore
b Kemerovo Institute of Food Science and Technology (University), Kemerovo, Russia
c Kemerovo State University, Kemerovo, Russia
Copyright ©M. et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0. (
http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Abstract
Lactoferrin (LTF) is a multifunctional protein of the transferrin family present in human and other mammals’ milk. Global demand for originated protein is currently much higher than the supply, and lactoferrin remains the one of the most expensive proteins. Therefore, the development of cost efficient methods to produce lactofferin is extremely important. In this study, human lactoferrin is obtained in bacterial system. ltf gene, coding sequence of protein - human lactoferrin (hLTF), was cloned into the expression vector pET28a+, modified signal sequence IMKKTAIAIAVALAGFATVAQA AS. E.coli BL21DE3 strain was selected for expression. Productivity of the producing strain in regard to recombinant human lactoferrin was 30-35% of the cellular protein. The strain produced the recombinant lactoferrin in the form of inclusion bodies. The maximum yield of the target protein (including in soluble form) was achieved by culturing of the recombinant strain at 25°C, induction by 1.1 mM concentration of IPTG and 8-h period after induction. Optimization of solubilization and renaturation conditions allowed once to recover lactoferrin in a concentration of 2.9 mg/ml by the pulsed system. The protein of high purity at least 90% was obtained by means of affinity chromatography on Ni+-agarose.
Keywords
lactoferrin,
milk protein,
gene,
E.coli
REFERENCES
- Patrick H.C., Welling M.M., and Geerts M. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nature biotechnology, 2014, vol. 34, pp. 484-487.
- Ostroumov L.A., Prosekov A.Y., Babich O.O., and Milenteva I.S. Usege of milk production waste for obtaining functional food products for baby and dietetic nutrition. European Journal of Natural History, 2013, no. 2, pp. 41-45.
- Hiroyuki W., Yamauchi K., and Takase M. Lactoferrin research, technology and applications. Int Dairy J., 2006, no. 11, pp. 1241-1251.
- Conesa C., Calvo M., and Sánchez L. Recombinant human lactoferrin: A valuable protein for pharmaceutical products and functional foods. Biotechnology Advances, 2010, no. 28, pp. 831-838.
- Veen H. Production and characterization of recombinant human lactoferrin. dissertation the degree of Doctor. Leiden, The Netherlands, 2008.
- Jiang T., Lijun Chen, Shiqian Jia, Lishui Chen, and Ying Ma. High-level expression and production of human lactoferrin in Pichia pastoris. Dairy Sci. Technol., 2008, vol. 88, pp. 173-181.
- Orla M. Conneely, Denis R. Headon, Bert W. O'Malley. Expression Of Processed Recombinant Lactoferrin And Lactoferrin Polypeptide Fragments From A Fusion Product In Aspergillus. Patent WO 1996/014413, 17.05.1997.
- Chong D.K. and Langridge W.H. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res., 2000, no. 9. pp. 71-78.
- Choi S.M., Lee O.S., Kwon S.Y., Kwak S.S., Yu D.Y., and Lee H.S. High expression of a human lactoferrin in transgenic tobacco cell cultures. Biotechnol Lett, 2003, vol. 25, pp. 213-218.
- Kwon S.Y., Jo S.H., Lee O.S., Choi S.M., Kwak S.S., and Lee H.S. Transgenic ginseng cell lines that produce high levels of a human lactoferrin, Planta Med., 2003, vol. 69, pp. 1005-1008.
- Suzuki Y.A., Kelleher S.L., Yalda D., Wu L., Huang J., Huang N., and Lönnerdal B. Expression, characterization, and biologic activity of recombinant human lactoferrin in rice, J Pediatr Gastroenterol Nutr., 2003, vol. 36, pp. 190-199.
- Li L., Shen W., Min L., Dong H., Sun Y., and Pan Q. Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer. Reprod Fertil Dev., 2006, no. 18, pp. 689-695.
- Jan H. Nuyens, Harry H. Van Veen. Isolation of lactoferrin from milk. Patent US 5919913, 6.07.1999.
- Goldman I.L. Production of human lactoferrin in animal milk. Biochem Cell Biol., 2012, no. 3, pp. 513-519.
- Babich O.O., Prosekov A.Y., Pokrovsky V.S., Sokolov N.N., and Anisimova N.Y. Recombinant l-phenylalanine ammonia lyase from rhodosporidium toruloides as a potential anticancer AGENT. Biotechnology and Applied Biochemistry, 2013, no. 3, pp. 316-322.
- Babich O.O., Soldatova L.S., and Prosekov A.Y. Cloning and expression of l-phenylalanine ammonia-lyase gene and characterization of its expression product. Biotechnology, 2011, no. 4, pp.33.
- Mergulhão F.J., Taipa M.A., Cabral J.M., and Monteiro G.A. Evaluation of bottlenecks in proinsulin secretion by Esherichia coli. J. Biotechnol., 2004, vol. 109, pp. 31-43.
- Nucleic Acid Isolation and Purification Manual, Roshe. 2010.
- Mini-PROTEAN® 3 Cell. Instruction Manual, Biorad. 2009.
