Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system

Protein Expression and Purification
Sheryl FernandesRajni Hatti-Kaul

Abstract

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 purificati...Continue Reading

References

Jun 1, 1986·Analytical Biochemistry·H Walter, G Johansson
Aug 1, 1986·Applied Biochemistry and Biotechnology·G Johansson, M Joelsson
Mar 10, 1998·European Journal of Biochemistry·T L ErikssonM van Hage-Hamsten
Apr 18, 2000·Journal of Colloid and Interface Science·A Millqvist-FurebyB Bergenståhl
May 4, 2001·Journal of Chromatography. B, Biomedical Sciences and Applications·P ArvidssonB Mattiasson
Jul 13, 2002·Biotechnology and Bioengineering·Sheryl FernandesBo Mattiasson
Jul 20, 1996·Biotechnology and Bioengineering·J CarlssonL Bülow

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Citations

Dec 15, 2004·Protein Expression and Purification·Beatrice-Elena DursinaKirill Alexandrov
Aug 9, 2013·Biotechnology Progress·João Vitor Dutra MolinoMaria Silvia Viccari Gatti

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