Abstract
A double mutant of rat trypsinogen (Asp189Ser, ΔAsp223) was constructed by site-directed mutagenesis. The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector. The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method. In case of this latter method the specificity profile of the enzyme was examined by simultaneous digestion of a mixture of oligopeptide substrates each differing only at the P 1 site residue, and the results were analyzed by high-performance liquid chromatography. All these assays unanimously demonstrated that the recombinant proteinase lacks trypsin-like activity but acquired a rather unique selectivity: it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues. This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing.
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