Generation of Mutant SNAP-23 to Arrest Mast Cell Degranulation at Trans-SNARE Complex Formation

Regulated secretion of pro-inflammatory molecules (e.g, histamines, proteases) from mast cells plays critical roles in immunity, allergic reactions, cardiovascular disease and cancer. These molecules are stored in secretory granules inside the cell and are rapidly released into the extracellular environment when mast cells are activated. It is known that mast cell degranulation depends upon membrane anchored SNAREs (soluble N-ethylmaleidimide-sensitive factor attachment protein receptors) and accessory proteins that form the trans-SNARE complex, a 4 helical bundle central to exocytic fusion. There are three SNARE proteins that contribute to the 4-helical bundle during exocytosis; Syntaxin and VAMP proteins each provide one helix each, SNAP-23 provides two helices. However, the biochemical properties of mast cell exocytosis are unclear because under physiological conditions the granules quickly proceed to fuse to the membrane. To investigate the properties of the degranulation related trans-SNARE complex, we decided to create a C-terminal truncation mutant of SNAP-23, which would arrest membrane fusion after the formation of the 4-helical bundle. Using PCR, SNAP-23 cDNA was amplified and inserted into pMBP-parallel expression vector; the ligation mixture was then transformed into E. coli cells and confirmed by sequencing. The wild type SNAP-23 was used as a template with custom made primers, amplified and inserted to yield a pMBP-parallel expression vector containing a mutant SNAP-23 cDNA lacking the C-terminal portion of the SNARE motif. The SNARE motif was sub-cloned and an IPTG induction process was used to induce to expression in Rosetta 2 E. coli cells. In this experiment, we were able to achieve successful cloning and expression of the mutant SNAP-23 omitting the c-terminus