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Affinity‐Specific protein separations using ligand‐coupled particles in aqueous two‐phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae
Author(s) -
Ku CheAn,
Henry Joseph D.,
Blair James B.
Publication year - 1989
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260330903
Subject(s) - pyruvate kinase , chemistry , alcohol dehydrogenase , aqueous solution , polyethylene glycol , aqueous two phase system , chromatography , ligand (biochemistry) , biochemistry , enzyme , organic chemistry , receptor , glycolysis
The novel approach of using aqueous two‐phase systems for the elution of protein from ligand‐coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G‐A‐coupled Sepharose CL6B (Blue‐Sepharose) particles as a model system. The ligand‐coupled particles distribute quantitatively to the polyethylene glycol‐(PEG‐) rich top phase and the recovered enzymes partition selectively to the dextran‐(DEX‐) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue‐Sepharose particles using PEG8000‐DEXT500 aqueous two‐phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two‐phase systems. Recovery of pyruvate kinase, which is bound to ligand‐coupled particles, in the DEX‐rich bottom phase of aqueous two‐phase systems can be up to 95% in one‐step operations. The mixing time of ligand‐coupled particles with aqueous two‐phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two‐phase systems also influence the recovery of pyruvate kinase from ligand‐coupled particles. The recovered enzymes in the DEX‐rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand‐coupled particles are added advantages of the process using aqueous two‐phase systems. Preliminary studies with goat polyclonal anti‐pyruvate kinase‐coupled Sepharose particles indicate that the process also may be applicable when a high‐affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.