The goal of the proposed project is to use structure based modeling and molecular biology techniques to increase trypsin and serine collagenase specificity for substrates with His residues at the P1 position. A variant of trypsin capable of utilizing a substrate assisted catalytic mechanism (Tn H57A) will be refined to increase its hydrolysis activity against substrates containing a His residue in the P1 position. Additionally, a His57=C6Ala variant of serine collagenase from the fiddler crab Uca pugilator will be expressed and tested against similar substrates. The serine collagenase variant is expected to have greater observable activity than the redesigned trypsin due to its unique substrate recognition attributes. The technology developed can be used to selectively cleave histidine tags from recombinant proteins.
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