SBNet

SBNet - Research Reports 1999

Jan Saras

Position: "Forskare" (UU)

Project: Structural studies on membrane proteins

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 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 and has been shown to interact with phosphatidylinositolphosphates with high affinity. Furthermore, Ezrin 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. In order to avoid precipitation of the FERM domain, a new protocol for thrombin digestion of the GST-FERM domain fusion protein has been established. The FERM domain is soluble up to 4-5 mg/ml. Major efforts are now focused on finding conditions that can promote the formation of protein crystals.

Budget.

This project is funded by SBNet (salary plus 50 kSEK consumables p.a.).


SBNet Latest update at 3 April, 2000.