HIS-TAG beta-GALACTOSIDASE IN HETEROGENOUS SYSTEM

FLORES, SS; SANCHEZ, JM; MOTTOLA, M; PERILLO, MA

Buenos Aires.

Otro; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades Argentinas de Biociencias

The Escherichia coli β-galactosidase (β-Gal) is a widely used enzyme for basic and applied research. Usually, the enzyme activity is measured in heterogeneous media. In our laboratory, we demonstrated that β-Gal can interact with lipid/water interfaces. In this condition we observed an enhanced catalytic activity, an increase in thermostability and the prevention of β-Gal proteolysis. Currently we are producing a recombinant β-GalHis ( 6 histidine residues at the carboxy terminus). These additional histidines (His-tag) decreases significantly (circa 50%) the catalytic activity of the protein. On the other hand, in the presence of lipid interfaces an important superactivation (up to 100%) is also observed. Since at the working pH 6.8 an important population of histidine residues are dissociated and conferred additional positive charges to the enzyme, we evaluated the ability of negatively charged interfaces to modulate β-GalHis activity. Interestingly the superactivation exerted by DOPC:DOPG (80:20, molar ratio) MLVs is even greater than that induced by zwitterionic bilayers of pure DOPC. The molecular organization of those lipid mixtures at the air-water interface served as membrane models and were studied through surface pressure?mean molecular area compression isotherms. Monolayers were composed of pure DOPC, pure DOPG or DOPC:DOPG mixtures at different molar ratios (80:20, 60:40, 40:60 and 20:80). Mixtures showed a non-ideal behavior with positive deviation from ideality (repulsive interaction between the components). Our results suggest that, beyond the attractive electrostactic interactions between the positively charged His tag and the negatively charged surface, the higher sensitivity to the negative interface is being favored by the reduction of the surface free energy associated to the β-GalHis?membrane binding. This contributes to an increased surface density of β-GalHis at the membrane which translates into an increased specific activity.