Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system.
Affinity extraction of dye- and metal ion-binding proteins, respectively, in a polyvinylpyrrolidone (PVP40)-Reppal PES 100 two-phase system was investigated. Due to the ability of PVP to complex azo dyes and inorganic ions, covalent coupling of the ligands was not essential. Cibacron Blue F3GA was used as the ligand for extraction of lactate dehydrogenase (LDH) from porcine muscle, while copper ions were used for extraction of B. stearothermophilus LDH with a fusion tag of six histidine residues (His6-LDH) from recombinant Escherichia coli homogenate. The binding strength of the enzymes to their respective ligands was only slightly reduced in the presence of PVP. The partition coefficient of Cibacron Blue and Cu2+ ions in the two-phase systems composed of different concentrations of PVP and Reppal was in the range of 20-30, with maximal partitioning being observed in the 17% (w/w) PVP40-10% Reppal PES100 system. Only a minor leakage of the ligands to the bottom phase was observed with time. The partitioning of porcine LDH to the PVP phase was increased 100-fold, and a maximal recovery of 89% was obtained in the two-phase system loaded with 0.2% (w/w) Cibacron Blue. The enzyme was quantitatively recovered with further purification from the PVP-dye phase using a secondary extraction step with 170 mM phosphate or alternatively with 100 mM phosphate containing NADH or NaCl. A more than 10-fold increase in the partition coefficient of His6-LDH was achieved in the two-phase system loaded with 0.4% (w/w) copper sulfate compared to the system lacking the metal ions. The enzyme was also back-extracted into phosphate phase in the presence of imidazole.
Fernandes
Sheryl
S
Department of Biotechnology, Center for Chemistry and Chemical Engineering, S-221 00 Lund, Sweden.
Kim
Hyung-Seog
HS
Hatti-Kaul
Rajni
R
D000818:Animals; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry; D019327:Copper Sulfate / Q000737:chemistry; D001411:Geobacillus stearothermophilus; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D009132:Muscles / Q000737:chemistry; D011205:Povidone / Q000737:chemistry*; D013213:Starch / Q000031:analogs & derivatives* / Q000737:chemistry*; D013552:Swine; D014227:Triazines / Q000737:chemistry
C103009:Reppal PES 100; D014227:Triazines; C012842:Cibacron Blue F 3GA; D003300:Copper; D013213:Starch; D007770:L-Lactate Dehydrogenase; D011205:Povidone; D019327:Copper Sulfate