Atlantic salmon (Salmo salar) expresses three trypsin isozymes; two anionic trypsins, Try-1 and Try-2, and a cationic trypsin, Try-3.  These trypsins are cold-adapted, or psychrophilic, enzymes possessing high catalytic efficiency at significantly lower temperatures than trypsins from mesophilic species.  Psychrophilic enzymes are typically more conformationally-flexible than mesophilic enzymes and have lower thermal stability.  The precise relationship between the dynamical properties of psychrophilic enzymes and their enhanced catalytic abilities at low temperatures is not fully understood. Some insight into this problem has been obtained from recent molecular dynamics simulations but testing the predictions of these theoretical studies will require access to site-specific mutants of  Salmo salar Try-1. Unfortunately, there are significant challenges to the heterologous expression and purification of eukaryotic psychrophilic enzymes, arising from such factors as their thermal inactivation at relatively low temperatures, aggregation, and autolysis. In an effort to express and purify tagged forms of recombinant Salmo salar Try-1, the gene for the inactive trypsinogen has been optimized for E. coli codon usage and engineered into a range of vectors for native expression and as modified forms, including thioredoxin and SUMO fusion proteins. This poster will describe our systematic efforts to develop a protocol to obtain high yields of this eukaryotic protein, together with a series of site-specific variants, using a variety of Escherichia coli expression host strains and conditions.

Development of an expression and purification system for Atlantic salmon (<i>Salmo salar</i>) anionic trypsin-1 (Try-1), a psychrophilic enzyme

246th National Meeting (2013)

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Development of an expression and purification system for Atlantic salmon (Salmo salar) anionic trypsin-1 (Try-1), a psychrophilic enzyme