Mutant rat trypsin selectively cleaves tyrosyl peptide bonds

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Protein Engineering

Background:

• Trypsinogen is a precursor to trypsin, a key digestive enzyme.
• Site-directed mutagenesis allows targeted modification of protein properties.
• Recombinant protein production in Escherichia coli is a standard biotechnological approach.

Purpose of the Study:

• To construct and characterize a double mutant of rat trypsinogen.
• To investigate the enzymatic activity and substrate specificity of the engineered protein.
• To explore potential applications in peptide analysis.

Main Methods:

• Site-directed mutagenesis to create Asp189Ser, DeltaAsp223 rat trypsinogen mutant.
• Recombinant protein expression in Escherichia coli using a periplasmic expression vector.
• Enzyme purification, enterokinase activation, and characterization using synthetic and natural substrates.
• Specificity profiling via simultaneous digestion of oligopeptide mixtures and high-performance liquid chromatography analysis.

Main Results:

• The recombinant double mutant proteinase demonstrated a complete lack of trypsin-like activity.
• The enzyme exhibited a unique substrate specificity, preferentially hydrolyzing peptide bonds C-terminal to tyrosyl residues.
• High-performance liquid chromatography analysis confirmed the specific cleavage pattern.

Conclusions:

• The engineered rat trypsinogen mutant possesses a novel, narrow substrate specificity.
• This tyrosyl-specific proteinase is a valuable tool for peptide-analytical applications.
• Potential uses include sequence-specific fragmentation of large proteins for subsequent sequencing.