Peptides as affinity surfaces for protein purification.
The tetrapeptide GPRP was previously shown to be an effective affinity ligand for fibrinogen when immobilized to Fractogel (Kuyas et al., 1990). The authors synthesized the GPRP peptide directly onto an aminefunctionalized POROS chromatographic resin to demonstrate the effectiveness of this approach for generating perfusive affinity media. Fibrinogen from plasma bound to an NH2-GPRP-POROS column under 50 mM phosphate buffer, 0.15 M NaCl, pH 7 at 15 ml/min flow rate. The bound fibrinogen showed weak clotting activity when eluted with 20 mM acetate buffer, pH 4. The peptide column did not bind denatured fibrinogen. The dynamic binding capacity of the column by frontal analysis was 10.2 mg/ml column volume. The total analysis time was under 5 min. Similarly, the CAQCHTVEK peptide of cytochrome c with heme group covalently attached to the SH groups of the two cysteines is known to bind to albumins (Adams et al., 1989). A simplified peptide analogue, GAQGHTVEK, was synthesized directlyon POROS resin. Under 20 mM MES, pH 6, albumin from human serum bound specifically to this peptide column and eluted with a salt gradient at 0.2 M NaCl, 20 mM MES (2-[N-Morpholino]ethane sulfonic acid), pH 6. The dynamic binding capacity of human serum albumin by frontal analysis was 19 mg/ml column volume. Thus, this column can purify albumin from human serum under non-denaturing conditions.
Pingali
A
A
PerSeptive Biosystems, Framingham, MA-01701, USA.
McGuinness
B
B
Keshishian
H
H
Jing
F W
FW
Varady
L
L
Regnier
F
F
D002846:Chromatography, Affinity / Q000379:methods*; D005340:Fibrinogen / Q000302:isolation & purification; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification*; D012116:Resins, Plant; D012709:Serum Albumin / Q000302:isolation & purification
D010455:Peptides; D011506:Proteins; D012116:Resins, Plant; D012709:Serum Albumin; D005340:Fibrinogen