Production of recombinant human lactoferrin in the allantoic fluid of embryonated chicken eggs and its characteristics.
The human iron-binding protein lactoferrin (hLf) has been implicated in a number of important physiological pathways, including those regulating immune function and tumor growth. In an effort to develop an efficient system for production of recombinant hLf (rhLf) that is structurally and functionally equivalent to the natural protein, we generated a recombinant CELO (chicken embryo lethal orphan) avian adenovirus containing an expression cassette for hLf. Embryonated chicken eggs were infected with the generated CELO-Lf virus. rhLf expression was measured in the allantoic fluid of infected eggs by ELISA three days later. The level of recombinant protein was about 0.8mg per embryo. rhLf was efficiently purified (up to 85% yield) from the allantoic fluid of infected eggs using affinity chromatography. rhLf produced in the allantoic fluid was characterized in comparison with natural hLf (nhLf) purified from human breast milk. SDS-PAGE, Western blotting and glycosylation analyzes showed that the recombinant protein had similar physical characteristics to nhLf. In addition, we demonstrated that the antioxidative and antimicrobial activity of rhLf produced in this system is equivalent to that of nhLf. Taken together, these results illustrate the utility of the described "recombinant CELO adenovirus-chicken embryo" system for production of functionally active rhLf. Efficient production of rhLf with accurate structure and function is an important step in furthering investigation of Lf as a potential human drug.
Tutykhina
Irina L
IL
Laboratory of Molecular Biotechnology, Gamaleya Research Institute of Epidemiology and Microbiology, 18, Gamaleya Street, Moscow 123098, Russia.
Bezborodova
Olga A
OA
Shmarov
Maxim M
MM
Logunov
Denis Y
DY
Neugodova
Galina L
GL
Nemtsova
Elena R
ER
Naroditsky
Boris S
BS
Yakubovskaya
Raisa I
RI
Gintsburg
Alexander L
AL
D000482:Allantois; D000818:Animals; D000259:Aviadenovirus; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002642:Chick Embryo; D015870:Gene Expression; D006801:Humans; D007781:Lactoferrin; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D015053:Zygote
D002352:Carrier Proteins; C490098:LTF protein, human; D011994:Recombinant Proteins; D007781:Lactoferrin