Rational Design of a Calcium-Independent Trypsin Variant

Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential based on its catalytic activity, substrate specificity, and robustness non-physiological reaction conditions. latter mainly due to stable protein fold, which six intramolecular disulfide bridges make significant contribution. Although trypsin does not depend cofactors, it essentially requires the binding of calcium ions calcium-binding site obtain complete enzymatic activity stability. This behavior inevitably associated with limitation enzyme’s applicability. To intrinsically calcium-independent, we removed native replaced another bridge. resulting stabilized apo-trypsin (aTn) retains full as proven by enzyme kinetics. Studies using Ellmann’s reagent further prove that two inserted cysteines at positions Glu70 Glu80 are their oxidized state, creating desired functional bond. Furthermore, aTn independent ions, possesses increased thermal stability, significantly reduced autolysis compared wildtype trypsin. Finally, confirmed our experimental data solving X-ray crystal structure aTn.