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N-Terminal Modification of Proteins with Subtiligase Specificity Variants
Amy M Weeks1,2, James A Wells1,3
1Department of Pharmaceutical Chemistry, University of California, San Francisco, California.
Current Protocols in Chemical Biology
|February 20, 2020
Summary
Subtiligase enables precise N-terminal protein modification by ligating peptide substrates. Newly developed mutants expand its utility for site-specific bioconjugation and cellular N-termini sequencing.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Subtiligase is an enzyme for N-terminal protein and peptide modification.
- It ligates peptide ester substrates to unblocked N termini, forming amide bonds.
- This enables site-specific introduction of chemical probes and payloads.
Purpose of the Study:
- To outline protocols for using subtiligase and its variants for protein modification.
- To detail methods for site-specific bioconjugation of purified proteins.
- To describe global modification of cellular N termini for sequencing.
Main Methods:
- Subtiligase-catalyzed N-terminal protein bioconjugation.
- Expression and purification of His-tagged subtiligase.
- Subtiligase substrate synthesis.
- Subtiligase N-terminomics using specificity mutants.
Main Results:
- A panel of subtiligase mutants expands substrate sequence compatibility.
- Protocols are provided for both purified protein and cellular N-terminal modification.
- The method allows site-specific attachment of diverse payloads.
Conclusions:
- Subtiligase is a versatile tool for protein engineering and N-terminomics.
- Mutant subtiligases enhance the scope and efficiency of N-terminal modifications.
- This technology facilitates advanced applications in chemical biology and proteomics.
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