<p>Stabilization of proteins is of great interest for the biotechnological society, industrial as well as research areas. Proteins with high stability are more suitable as reagents, easier to handle, store, transport and use in industrial processes. One way to stabilize a protein is to introduce a disulfide bridge into the structure by protein engineering. In this report the formation of a disulfide bridge between engineered cysteines in non ideal conformations in human carbonic anhydrase has been investigated. The disulfide bridge is not formed when the protein is in its native state. It is shown that when the protein is exposed to mild concentrations of urea in the presence of DTTox the disulfide bridge is formed. Also upon refolding in vitro, in a non oxidative environment, disulfide bridges are formed. This observation is worth to notice, since the disulfide bridge does not form to any appreciable extent when the protein is expressed and folded in vivo in Escherichia coli.</p>

Stabilization of proteins is of great interest for the biotechnological society, industrial as well as research areas. Proteins with high stability are more suitable as reagents, easier to handle, store, transport and use in industrial processes. One way to stabilize a protein is to introduce a disulfide bridge into the structure by protein engineering. In this report the formation of a disulfide bridge between engineered cysteines in non ideal conformations in human carbonic anhydrase has been investigated. The disulfide bridge is not formed when the protein is in its native state. It is shown that when the protein is exposed to mild concentrations of urea in the presence of DTTox the disulfide bridge is formed. Also upon refolding in vitro, in a non oxidative environment, disulfide bridges are formed. This observation is worth to notice, since the disulfide bridge does not form to any appreciable extent when the protein is expressed and folded in vivo in Escherichia coli.