A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties.
A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.
Wu
Lin
L
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China.
Wu
Jinhui
J
Zhang
Jian
J
Zhou
Yuanyuan
Y
Ren
Guoyan
G
Hu
Yiqiao
Y
D000818:Animals; D001769:Blood; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014168:Transferrin / Q000032:analysis* / Q000302:isolation & purification
D014168:Transferrin