Refolding of Carbonic Anhydrase Assisted by 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Liposomes

Abstract

The interaction between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposome and denatured bovine carbonic anhydrase (CAB; EC 4.2.1.1) was investigated with the aim of developing an effective liposome-assisted protein refolding process. The liposome-denatured CAB interaction facilitated the recovery of CAB activity. In the presence of liposomes ([POPC] = 0.25 mM), 78 ± 7% of guanidinium hydrochloride (GuHCl)-denatured CAB was reactivated at 25 °C (final concentrations of CAB and GuHCl were 3.3 μM and 0.1 M, respectively), while the refolding yield reached only 50 ± 5% in the absence of liposomes. Furthermore, the enzymatic activity of refolded CAB was maintained for at least 1.5 h if the CAB refolding operation was carried out in the presence of liposomes even at high temperature (∼55 °C). The data obtained can be interpreted as follows. The first step in this liposome-assisted CAB refolding is the binding of a refolding intermediate of CAB to the outer surface of liposomes mainly due to hydrophobic interaction. At this stage, the formation of inactive intermolecular aggregates of CAB was prevented by the interaction with the liposomes. Subsequently, the native refolded CAB molecules were released spontaneously from the liposome-CAB complexes. In the liposome-assisted CAB refolding process, the reactivation yield of CAB was affected by the physical properties of the liposome membrane. Surface hydrophobicity and fluidity of liposomes, which were dependent on liposome size and temperature, were found to be key parameters for the improvement of the reactivation yield of CAB.