N-terminal cysteinyl proteins can be prepared using thrombin cleavage

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Expressed protein ligation (EPL) is a powerful technique for joining native proteins via a peptide bond in aqueous solution.
• EPL requires specific protein modifications: a C-terminal alpha-thioester and an N-terminal cysteine (Cys).
• Current methods for generating N-terminal Cys often rely on Factor Xa or TEV proteases.

Purpose of the Study:

• To introduce thrombin as a novel protease for generating N-terminal Cys residues.
• To provide an alternative reagent for expressed protein ligation.
• To demonstrate the utility of thrombin in protein engineering and bioconjugation.

Main Methods:

• Engineered protein expression vectors with a modified thrombin cleavage site (LVPRG to LVPRC).
• Utilized thrombin protease for enzymatic cleavage to yield N-terminal Cys.
• Applied the method to produce N-terminal Cys on Csk and Abl tyrosine kinase domains.

Main Results:

• Successfully generated proteins with N-terminal Cys using thrombin protease.
• Demonstrated the applicability of this method on two distinct protein domains.
• Showcased the convenience of vector modification for thrombin-mediated cleavage.

Conclusions:

• Thrombin protease is an effective and versatile reagent for generating N-terminal Cys for expressed protein ligation.
• This method expands the toolkit for protein ligation, overcoming limitations of existing proteases.
• The approach holds potential for broader biotechnological applications requiring precise protein conjugation.