Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

Steinhagen, Max; Zunker, Katja; Nordsieck, Karoline; Beck‐Sickinger, Annette G.

doi:10.1016/j.bmc.2013.03.039

Cited by 28 publications

(24 citation statements)

References 48 publications

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“…It is well established that the removal of reaction by-products or residual sortase enzyme can be achieved through the strategic inclusion of His 6 affinity handles, or the use of the elastin-like polypeptide as a controlled solubility switch [3, 26, 27, 31]. SrtA staph has also been covalently immobilized on sepharose or PEGA resins to facilitate enzyme removal and enzyme recycling [14, 32]. A useful extension of sortase-immobilization has been the construction of flow-based systems in which reactants are passed over immobilized sortase columns [14, 33].…”

Section: Optimizing Srtastaph Performancementioning

confidence: 99%

Recent advances in sortase-catalyzed ligation methodology

Antos

Truttmann

Ploegh

2016

Current Opinion in Structural Biology

144

139

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The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology. Topics include the use of engineered sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a sortase-based approach.

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Section: Optimizing Srtastaph Performancementioning

confidence: 99%

Recent advances in sortase-catalyzed ligation methodology

Antos

Truttmann

Ploegh

2016

Current Opinion in Structural Biology

144

139

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“…The antibodies to be joined can be expressed separately as full-length antibodies, and the coupling reactions occur under physiological conditions. Likewise, sortase-catalyzed reactions enable largescale preparation of modified biomolecules (49) and immunotoxins (50). A panel of sortase-modified antibodies equipped with click handles far more readily facilitates combinatorial exploration of many different combinations of C-C-linked bispecific antibodies than genetic fusions would allow.…”

Section: Discussionmentioning

confidence: 99%

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

Wagner

Kwakkenbos

Claassen

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance Bispecific antibodies expand the function of conventional antibodies. However, therapeutic application of bispecifics is hampered by the reduced physiochemical stability of such molecules. We present a format for bispecific antibodies, fusing two full-sized antibodies via their C termini. This format does not require mutations in the antibody constant domains beyond installation of a five-residue tag, ensuring that the native antibody structure is fully retained in the bispecific product. We have validated the approach by linking two anti-influenza A antibodies, each active against a different subgroup of the virus. The bispecific antibody dimer retains the activity and the stability of the two original antibodies.

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“…Detailed protocols for production of sortases, sortase substrate design, peptide synthesis, and sortase reactions in solution are provided in previous protocols 20,21 . Sortase A immobiliziation has been previously described using a different strategy than detailed in this protocol 22 . The protocol outlined here offers a more rapid, covalent immobilization procedure, as well as the use of the calcium-independent heptamutant sortase A to increase the utility of the reaction.…”

Section: Advantages Of Sortase-mediated Reactions Using Immobilized Smentioning

confidence: 99%

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

Witte

Guimarães

et al. 2015

Nat Protoc

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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, bioorthogonal 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. Small 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 used for the modification of calcium-senstive substrates or in instances where low temperatures are needed. Preparation of immobilized sortase A takes 1–2 days. Batch reactions take 3–12 hours and flow reactions proceed at 0.5 mL per hour, depending on the geometry of the reactor used.

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Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

Cited by 28 publications

References 48 publications

Recent advances in sortase-catalyzed ligation methodology

Recent advances in sortase-catalyzed ligation methodology

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

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

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