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Reprogramming the specificity of sortase enzymes.

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Researchers engineered new versions of Staphylococcus aureus sortase A, an enzyme used for protein modification. These engineered sortases (sortase A variants) can now recognize altered peptide substrates, enabling precise, simultaneous labeling of multiple proteins.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Protein Engineering
  • Enzymology

Background:

  • Staphylococcus aureus sortase A is a transpeptidase crucial for site-specific protein modification.
  • Its stringent substrate specificity limits its application in complex biological systems.
  • There is a need for orthogonal sortase variants to expand protein labeling capabilities.

Purpose of the Study:

  • To evolve orthogonal sortase A variants with altered substrate specificities.
  • To enable simultaneous, site-specific conjugation of multiple peptide substrates.
  • To demonstrate the utility of engineered sortases in complex biological samples and therapeutic protein synthesis.

Main Methods:

  • Laboratory evolution of sortase A using yeast display screening.
  • Integration of nine rounds of screening with negative selection.
  • Characterization of evolved variants for substrate specificity and catalytic activity.

Main Results:

  • Developed two orthogonal sortase A variants recognizing LAXTG and LPXSG substrates.
  • Achieved specificity changes up to 51,000-fold compared to wild-type sortase A.
  • Demonstrated high specificity for target substrates (up to 24-fold) with retained catalytic activity.
  • Enabled simultaneous conjugation of multiple peptide substrates in a single solution.

Conclusions:

  • Engineered sortase A variants overcome the limitations of wild-type enzyme specificity.
  • Orthogonal sortases facilitate complex protein modifications, including labeling of endogenous proteins and synthesis of therapeutic protein conjugates.
  • These tools significantly advance the field of site-specific protein engineering and bioconjugation.