Interaction of human lactoferrin with DNA: one-step purification by affinity chromatography on single-stranded DNA-agarose.
Human lactoferrin has been purified to apparent homogeneity with high recovery (greater than 95%) in one chromatographic step using immobilized single-stranded DNA-agarose. Human colostral whey, at neutral pH, was loaded onto columns of DNA-agarose; a single purified protein, lactoferrin, was eluted using elevated ionic strength buffers. The lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, and reverse-phase (C18) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels and silver staining showed the purified lactoferrin preparation to contain a single protein of 78 kD with intact immunologic determinants. Similar results were obtained before and after iron saturation of the colostral whey proteins. Apolactoferrin purified in this manner was shown to bind iron with high affinity. These results demonstrate the effectiveness of immobilized DNA as one of the most rapid and complete lactoferrin purification procedures described thus far.
Hutchens
T W
TW
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Magnuson
J S
JS
Yip
T T
TT
D002846:Chromatography, Affinity / Q000379:methods*; D003126:Colostrum / Q000032:analysis; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007781:Lactoferrin / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000302:isolation & purification*; D011247:Pregnancy; D012685:Sepharose
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007782:Lactoglobulins; D012685:Sepharose; D007781:Lactoferrin