@conference{
author = "Četić, Danilo and Miljuš, Goran and Dobrijević, Zorana and Gligorijević, Nikola and Vilotić, Aleksandra and Nedić, Olgica and Penezić, Ana",
year = "2023",
abstract = "Human transferrin (Tf) is a bilobal 76 kDa iron-binding glycoprotein present in human
serum. Each lobe has the ability to bind one ferric ion (Fe3+) and a single synergistic
bicarbonate anion. The main role of Tf is to transport Fe3+ ions through the circulation to
cells, via interaction with transferrin receptor (TFR) on the cell surface. Previously
described methods for Tf isolation and purification are either very time-consuming or
provide Tf of lower final purity. Here we describe a fast and simple FPLC method for the
isolation and purification of Tf from human serum. Serum samples were prepared by
precipitation, while protein purification was performed on FPLC system, using an anionexchange
column. Several different buffers at the same pH were tested. Tf purified by this
method was analyzed by Western blot, followed by immunodetection, as well as with
silver staining after SDS PAGE. Its functionality was tested with respect to iron-binding
capacity (ferozzine method) and its ability to interact with TFR by immunofluorescent
staining. The conformation of purified Tf was analyzed by recording intrinsic fluorescent
emmision spectra originating from Trp residues. The method itself is highly reproducible
(intra- and interday), easy to perform (only two steps) and fast (under an hour), yielding
98% to 99% pure Tf with all buffers. Purified Tf was shown to have retained its ironbinding
capacity, as well as the ability to interact with TFR. Purified Tf also retained its
native three-dimensional structure. Described method for the isolation and purification of
Tf is fast, simple and highly reproducible, yielding a functional Tf of high purity in its
native state while offering the flexibility of using different buffer systems. All of these
features make this protocol a method of choice for the isolation and purification of Tf on a
semi-preparative scale.",
publisher = "Serbian Biochemical Society",
journal = "Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia",
title = "Simple two-step semi-preparative isolation and purification of transferrin from human serum",
pages = "66-66",
url = "https://hdl.handle.net/21.15107/rcub_cer_6663"
}
