Roxana Mesías

Roxana Mesías

Wheaton College



Mentor: Dr. Lisa Clark, Department of Microbiology

Sub-cloning of the leucine-rich extracellular domain of the human Toll-like receptor 4 gene into the pET23 expression plasmid

Toll-like receptors (TLRs) are a family of recognition receptors occurring on cells of the immune system that play a key role in pathogen recognition and initiation of inflammatory immune responses (IVIS, 2004). For example, cells such as macrophages that function to engulf and destroy invading bacteria and viruses, recognize and bind to these pathogens through their TLRs (Medzhitov and Janeway, 2002). All Toll-like receptors contain an extracellular protein-protein interaction domain known as the leucine-rich repeat (LRR) motif. Thus, TLRs are part of the LRR protein superfamily, a diverse group of proteins that mediate binding interactions in a wide variety of biological processes.

Mutations of LRR proteins and abnormal LRR protein expression have been linked to a variety of human diseases including cancer (Almeida et al., 1998), Crohn’s disease (Hugot et al., 2001) and Bernard-Soulier syndrome, a rare bleeding disorder (Ulsemer et al., 2000). Therefore, the discovery of new therapeutic drugs that could potentially block or enhance protein-protein binding interactions of key relevance to human health, such as those mediated by TLRs, is of fundamental importance. The identification of short amino acid sequences (peptides) involved in mediating binding at the molecular level is an important strategy in drug design (Bartfai et al., 2003). By mimicking these peptides with reagents that target specific binding surfaces on protein molecules, protein-protein interactions that may contribute to diseases could potentially be blocked. The overall goal of this investigation is to characterize the specific binding interactions of the Toll-like Receptor 4 (TLR4) protein with its binding partners (ligands) at the molecular level. For this study, the extracellular LRR domain of the TLR4 gene will be sub-cloned into an expression vector. This is an important step in the generation of large quantities of the TLR4-LRR binding protein required to carry out this investigation.

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