Semisynthesis of cytotoxic proteins using a modified protein splicing element

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Traditional methods for producing complex proteins can be challenging.
• Cytotoxic proteins like RNase A and HpaI have therapeutic and research applications.
• Intein-mediated protein ligation offers a potential solution for protein synthesis.

Purpose of the Study:

• To develop and validate a novel semisynthetic approach for producing cytotoxic proteins.
• To demonstrate the efficacy of intein-mediated ligation for generating full-length, active enzymes.
• To explore the potential for incorporating unnatural amino acids using this method.

Main Methods:

• Utilized a semisynthetic strategy involving protein splicing elements (inteins).
• Generated reactive C-terminal thioesters on truncated recombinant proteins (RNase A and HpaI).
• Ligated synthetic peptides to truncated proteins via nucleophilic attack, forming native peptide bonds.

Main Results:

• Successfully produced full-length bovine pancreatic ribonuclease A (RNase A) and HpaI restriction endonuclease.
• Ligated proteins exhibited catalytic activity comparable to wild-type enzymes.
• RNase A demonstrated turnover numbers and Km values consistent with literature.
• HpaI showed specific activity within the expected range.

Conclusions:

• The novel semisynthetic ligation technique is effective for producing active cytotoxic proteins.
• This method provides a robust platform for protein engineering, including the insertion of unnatural amino acids.
• The approach offers advantages for producing complex or modified proteins.