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

Abstract

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.

Recent advances in sortase-catalyzed ligation methodology

“…The pentamutant was subsequently enhanced by adding two additional mutations known to eliminate Ca 2+ dependency [12]. Two SrtA staph heptamutants have been described which add either E105K/E108A mutations or E105K/E108Q mutations to the SrtA staph pentamutant backbone [13•, 14, 15•]. These variants no longer require Ca 2+ , and also retain much of the enhanced rate characteristics of the SrtA staph pentamutant.…”

“…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].…”

“…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]. The flow-based reactor described by Pentelute and coworkers is particularly noteworthy in that the authors demonstrated that flow-based sortagging increased isolated product yields, and reduced contamination by hydrolytic, cyclic, or oligomeric by-products relative to analogous reactions performed using a standard solution-phase protocol [33].…”

Irreversible Protein Labeling by Paal–Knorr Conjugation

“…Third, probes containing warhead 10 are not reactive to media like many of the other functional handles, such as acrylates, α-haloamides, succinimidyl esters, or sulfonyl halides; an issue that often creates significant batch-based inhomogeneity, which has recently been addressed in part with continuous-flow reactors. [27] Finally, the fact that 1,4-diketones are resistant to metabolism [28] and have demonstrated Paal–Knorr reactivity in humans [14] indicate their potential for further advance as next generation tools for chemical biological applications.…”

Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments

“…We used a nucleophile of the sequence GGGK(TAMRA)K(azide) ( Figure S2). We used a mutant sortase with increased activity and Ca 2+ independence [17] . We achieved full conversion of VHH7 to the desired product with a single TAMRA dye and an azide handle for a "click" ligation.…”