PI: Mowbray/Jones (SLU/UU)
Project: Structural studies on membrane protein receptors
Background
The aim of the project is to determine the three dimensional structure of a member of the Band 4.1 superfamily, both with and without the physiologically relevant ligands. Proteins of this family are localised at the intracellular side of the plasma membrane of animal cells. These proteins are found in a number of cell adhesion structures and are thought to connect the cytoskeleton to the plasma membrane. Moreover, proteins of the Band 4.1 superfamily has recently been shown to be involved in signal transduction pathways, indicating that these proteins have complex functions involving both structural and signalling properties. Determination of the three-dimensional structure of members of this superfamily is important for the understanding of their physiological function.
One of the most well studied proteins in the Band 4.1 superfamily is Ezrin. The amino terminal FERM domain (FERM stands for the proteins Band 4.1, Ezrin, Radixin and Moesin) of Ezrin has been shown to be localised at the plasma membrane where it probably interacts with phosphatidylinositolphosphates with high affinity. Furthermore, Ezrin also interacts with the intracellular portion of the transmembrane proteins ICAM1, ICAM2, ICAM3 and CD44.
Present work
The cDNA coding for the FERM domain of Ezrin was cloned into a bacterial expression vector. The FERM domain is expressed as a glutathione S-transferase (GST) fusion protein that allows efficient purification of the protein using glutathione sepharose. Much work has been spent on optimising the expression conditions, since most of the expressed protein was initially found in the insoluble fraction. Currently, 1-2 mg soluble recombinant protein per litre culture can be obtained. A protocol for efficient removal of the GST fusion part, by using the protease thrombin, has been developed. The affinity purified FERM domain can be further purified to high purity with an ion exchange column. At high concentrations, the FERM domain tends to precipitate during the thrombin cleavage. Therefore, efforts are now focused of how to avoid this problem.
In order to study the activity of the recombinantly expressed FERM domain a binding assay is needed. The assay is based on the interaction between the FERM domain and the intracellular parts of ICAMs. The assay is not yet working satisfactory, however, the problem can most likely be overcome by using peptides of higher quality.
Latest update at 18 February, 1999.