Functional expression of mammalian receptors and membrane channels in different cells.

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other.

The 4.5 A structure of human AQP2.

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorption in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography.

Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography.

Large-scale purification of functional recombinant human aquaporin-2.

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system.

Gecko iota-crystallin: how cellular retinol-binding protein became an eye lens ultraviolet filter.

Eye lenses of various diurnal geckos contain up to 12% iota-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation.