Generation of a fusion protein containing the two functional coiled-coil domain of t- SNARE, SNAP-23 and a transmembrane domain for mast cell

Abstract

SNAREs (Soluble N-Ethylmaleimide-Sensitive Fusion Protein Attachment Protein Receptor) are a class of membrane proteins that mediate membrane-membrane fusion in eukaryotic cells. SNAP-23 is a t-SNARE which is a component of cellular machinery is required for membrane fusion. SNAP-23 lacks transmembrane domain. Cysteines in the linker region of SNAP-23 are involved in targeting of SNAP-23 to the membrane. In the present work, a portion of MDR3 gene (MDR3 1-145) and CLP24 (CLP134-195) was subcloned into a plasmid encoding EGFP-SNAP-23 Cys- mutant for the generation of a fusion protein containing the two functional coiled-coil domain of t-SNARE, SNAP 23 and a transmembrane domain of MDR3 gene and CLP24 for mast cell. This fusion protein will be important to study the membrane targeting and raft association of the chimeric SNAP23 protein, which plays an important role in mast cell exocytosis in the mammalian system. A novel bioinformatics approach has been applied to identify the specific transmembrane domain. This novel approach can be used to construct other fusion proteins.