Site-specific protein modification using immobilized sortase in batch and continuous-flow systems
Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describe...
| Main Authors: | , , , , , , , , , |
|---|---|
| Other Authors: | , |
| Format: | Article |
| Language: | English |
| Published: |
Springer Nature,
2017-03-07T19:21:07Z.
|
| Subjects: | |
| Online Access: | Get fulltext |
| Summary: | Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca[superscript 2+]-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h[superscript −1], depending on the geometry of the reactor used. National Institutes of Health (U.S.) (NIH grant no. RO1 AI087879) Netherlands Organization for Scientific Research (NWO) |
|---|