The expression and analysis of tetraspanin large extracellular domains
There are 33 tetraspanin proteins in mammals and these are Widely expressed on the majority of cells and tissues. Tetraspanin proteins are differentiated from other tetraspan proteins by a number of conserved motifs, in particular an absolutely conserved Cys- Cys-Gly motif in the large extracellular loop (EC2). This EC2 domain is the most variable region between tetraspanin proteins in length and amino acid sequence, and consequently is the most intriguing domain. Soluble recombinant EC2 domains have been used to study tetraspanin function, and so one of the main aims of this work was to optimise the production of GST-EC2 proteins from CD9, CD81, CD63, CD151. Methods were developed to produce these domains in large quantifies, and they were shown to have biological activity in the inhibition of giant cell formation in human monocytes. The soluble EC2 domains from CD63, CD9, CD81 and CD151 were all shown to potently inhibit HIV-1 infection of macrophages. Infection of the two major HIV-1 strains R5 and X4 was prevented by all the tetraspanins tested, suggesting an involvement of a tetraspanin enriched microdomain. The inhibition was macrophage specific since infection of PBMC was not prevented. ' Another aim was to clone and express EC2 domains from CD82 and CD231. CD82 and CD231 EC2 domains are larger and consequently proved more difficult to express. Both were cloned successfully into the pGEX-2T expression vector and CD82 was expressed in Origami cells (Novagen). CD82 EC2 was considered to be a valuable tool to make because of the involvement of CD82 in tumor cell metastasis. The original aim of the work was to characterise the binding between tetraspanin EC2 domains and primary ligands. However, no appropriate ligands were found. We did show by ELISA that mouse, but not human, CD9 bound to murine PGS17 but we could not detect the reported binding between CD9 and fibronectin. Additionally, efforts were made to clone and express two immunoglobulin-like proteins that had recently been identified as CD9 and CD81 primary binding partners. The cloning of full length EWI DNA as well and double Ig domains was carried out successfully into four different expression vectors, however, no protein expression was'detected in eukaryotic and a range of prokaryotic expression systems. Structural studies were performed using CD63 EC2. Strategies to cleave GST and to purify the EC2 domain were successfully developed. Instability problems of CD63 EC2 in the absence of GST did not allow for concentration of the cleaved product to levels required for crystal trials. CD63 EC2-His6 recombinant protein was used in X- ray crystallography studies, and over 1000 crystallisation conditions were tested in preliminary structural studies.