The Open Analytical Chemistry Journal

2010, 4 : 27-34
Published online 2010 July 16. DOI: 10.2174/1874065001004010027
Publisher ID: TOACJ-4-27

Comparison of Purification Efficiencies of Different Affinity Separation Techniques

Matthias Koetke , Henning Rosenfeld , Stephanie Peper and Bernd Niemeyer
Institute for Coastal Research/Marine Bioanalytic Chemistry, GKSS Research Centre, Max- Planck-Strasse, D-21502 Geesthacht, Germany.

ABSTRACT

An affinity separation method for the isolation of recombinantly produced benzoylformiate decarboxylase (BFD) is presented based on the interaction of the enzyme to its cofactor. Enzyme/cofactor interactions are well suited for affinity purification processes with regard to its high affinity and selectivity. Problems can be induced due to harsh desorption conditions that probably diminish the activity of the purified enzyme. The implementation of boronic acids to immobilize the cofactor introduces a gentle release by a simple pH shift from 8.5 to 6. The disadvantages of maminophenyl boronic acids are the high nonspecific adsorption induced by the introduction of a positive charge and a hydrophobic phenyl ring. It was shown that the disadvantages prevail and boronic acid resins are not suitable for protein purification from complex matrices. In contrast to the affinity separation method, where the cofactor was directly immobilized onto the support, a better separation performance could be achieved, which is reflected by a purification factor of 4.