Proteoliposomes in nanobiotechnology

Abstract

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

Keywords: Alkaline phosphatase; Biomineralization; Biotechnology; Leishmaniasis; Na,K-ATPase; Nanosensor; Proteoliposomes.

Fig. 1

Scheme for the obtention of a biomimetic system. Microscopic details: tissues or cell cultures can be used for the obtention of cells and, after an osmotic process, organelles or membrane fractions can be isolated by differential centrifugation. Molecular details: each protein can be solubilized and purified by a different method depending on its interaction with the lipid membrane (for details, see text). After solubilization and purification, the protein can be reconstituted in the membrane using direct insertion or co-solubilization processes (for details, see text). Alternatively, the protein can also be sequestered into the liposome

Fig. 2

Effect of increasing concentrations of ATP on the P_i-generating activity of osteoblast-derived TNAP: a membrane-bound; b reconstituted in DPPC:DODAB liposomes (8:2, molar ratio, o) and (9:1, molar ratio, ●); c reconstituted in DPPC liposomes and d reconstituted in DPPC:DPPS liposomes (8:2, molar ratio, o) and (9:1, molar ratio, ●). Assays were done at 37°C in 50 mM AMPOL buffer, pH 9.5, containing substrate and 2 mM MgCl₂ and released P_i measured

Fig. 3

Effect of increasing concentrations of PP_i on the P_i-generating activity of osteoblast-derived TNAP: a membrane-bound; b reconstituted in DPPC:DODAB liposomes (8:2, molar ratio, o) and (9:1, molar ratio, ●); c reconstituted in DPPC liposomes and d reconstituted in DPPC:DPPS liposomes (8:2, molar ratio, o) and (9:1, molar ratio, ●). Assays were done at 37°C in 50 mM AMPOL buffer, pH 9, containing substrate and 2 mM MgCl₂ and released P_i measured

Fig. 4

Scheme of the different approaches to use proteoliposomes: to study lipid–lipid, lipid–protein, and lipid-peptide interactions as delivery systems, as vesicular mimetic systems, as nanosensors or vaccines, and as a system to study interfaces