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

Martin D. Witte, Tongfei Wu, Carla P. Guimaraes, Christopher S. Theile, Annet E. M. Blom, Jessica R. Ingram, Zeyang Li, Lenka Kundrat, Shalom D. Goldberg, Hidde L. Ploegh*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

41 Citaten (Scopus)

Samenvatting

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 Ca2+-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(-1), depending on the geometry of the reactor used.

Originele taal-2English
Pagina's (van-tot)508-516
Aantal pagina's9
TijdschriftNature protocols
Volume10
Nummer van het tijdschrift3
DOI's
StatusPublished - mrt.-2015
Extern gepubliceerdJa

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