Mutant rat trypsin selectively cleaves tyrosyl peptide bonds

• Published in: Analytical biochemistry 2004-03, Vol.326 (2), p.190-199
• Main Authors: Pál, Gábor, Patthy, András, Antal, József, Gráf, László
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic Vectors - genetics; Limited proteolysis; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments - biosynthesis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide sequencing; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Substrate Specificity; Trypsin - chemistry; Trypsin - genetics; Trypsin - metabolism; Trypsinogen - biosynthesis; Trypsinogen - chemistry; Trypsinogen - genetics; Tyrosyl peptide bond cleavage
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.12.001

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.