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Site-specific protein modification using immobilized sortase in batch and continuous-flow systems.

Martin D Witte¹, Tongfei Wu², Carla P Guimaraes¹

¹Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

Nature Protocols

|February 27, 2015

Summary

This summary is machine-generated.

Immobilizing sortase A enzyme on beads simplifies protein modification and purification. This reusable, immobilized enzyme enables efficient site-specific labeling with various functional groups, including for calcium-sensitive applications.

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Area of Science:

Biochemistry
Protein Engineering
Enzyme Immobilization

Background:

Sortase A enzyme facilitates site-specific protein modification with diverse functional groups.
Current methods may involve complex purification steps after protein labeling.

Purpose of the Study:

To describe a protocol for immobilizing sortase A onto Sepharose beads.
To simplify downstream purification and enable enzyme reuse in protein modification reactions.

Main Methods:

Immobilization of sortase A (including a Ca(2+)-independent heptamutant variant) onto Sepharose beads.
Application of immobilized enzyme in batch and continuous-flow protein labeling reactions.
Characterization of reaction times and enzyme reusability.

Main Results:

Immobilized sortase A allows for simplified purification of site-specifically labeled proteins.
The immobilized enzyme can be reused for multiple modification cycles, reducing enzyme requirements.
The protocol is effective with both wild-type and a Ca(2+)-independent sortase A variant.

Conclusions:

Immobilizing sortase A on a solid support offers an efficient and reusable platform for protein modification.
This method streamlines the preparation of homogeneously modified proteins for various applications.
The protocol supports both standard and specialized applications, including those requiring calcium independence or low temperatures.