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Site-specific protein labeling via sortase-mediated transpeptidation
Maximilian Wei-Lin Popp1,2, John M Antos1, Hidde L Ploegh1,2
1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.
Current Protocols in Protein Science
|April 15, 2009
Summary
This study introduces a method for creating protein bioconjugates using sortase A enzyme. This enzyme enables site-specific attachment of functional probes to proteins under mild conditions, applicable both in solution and on cell surfaces.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Developing functional protein bioconjugates requires specific and mild methods for probe attachment.
- Sortase A, a transpeptidase from Staphylococcus aureus, offers a potential solution for site-specific protein modification.
Purpose of the Study:
- To describe a method for site-specific protein functionalization using sortase A.
- To demonstrate the utility of sortase A in creating protein bioconjugates with diverse probes.
Main Methods:
- Engineering target proteins with a sortase recognition sequence (LPXTG).
- Utilizing purified sortase A enzyme to cleave the recognition sequence.
- Conjugating functionalized oligoglycine peptides to the target protein via amide linkage.
Main Results:
- Achieved site-specific incorporation of reporters into target proteins.
- Demonstrated applicability of the method to proteins in solution.
- Showcased the method's effectiveness on living cell surfaces.
Conclusions:
- Sortase A-mediated ligation provides a versatile and efficient strategy for protein bioconjugation.
- This method facilitates the site-specific attachment of various functional probes under mild conditions.
- The technique is valuable for applications in both in vitro and in vivo protein modification.
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