A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk, one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge.
Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.
Holm
Jan
J
Department of Clinical Chemistry, Central Hospital, Herning, DK-7400, Denmark. BCMM-JH@BORA.DK
Hansen
Steen Ingemann
SI
Høier-Madsen
Mimi
M
D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002412:Cations; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004563:Electrochemistry; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008895:Milk, Human / Q000737:chemistry*; D011956:Receptors, Cell Surface
D002352:Carrier Proteins; D002412:Cations; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D008024:Ligands; D011956:Receptors, Cell Surface