Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

Tsukiji, Shinya; Nagamune, Teruyuki

doi:10.1002/cbic.200800724

Cited by 190 publications

(181 citation statements)

References 96 publications

(131 reference statements)

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“…Sortase A-catalyzed transpeptidation has emerged as a powerful tool for bioconjugation because of the enzyme's high specificity for the LPXTG motif and its extremely broad substrate tolerance outside of the recognition elements described above. Because the LPXTG and oligoglycine motifs can be flanked by virtually any biomolecule, sortase has been used to label proteins, generate nucleic acid-protein conjugates, and immobilize proteins onto solid supports (23). A significant limitation of srtA is the large quantities of the enzyme or long reaction times that are needed to overcome its poor reaction kinetics (k cat ∕K m LPETG ¼ 200 M −1 s −1 ; Table 1).…”

Section: Resultsmentioning

confidence: 99%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

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

511

584

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The ability to routinely generate efficient protein catalysts of bondforming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.

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Section: Resultsmentioning

confidence: 99%

A general strategy for the evolution of bond-forming enzymes using yeast display

Chen

Dorr

Liu

2011

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

511

584

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“…A site-specific method for labeling the N termini of cell surface proteins by sortase-A was recently developed (Fig. 7B) (16,17). By using this technique, we specifically labeled cell surface PAFRs in HeLa cells with Alexa Fluor 488 and compared translocation of the labeled P247A induced by mc-PAF with that of the WT by using confocal microscopy.…”

Section: Deficiency In Cell Surface Expression Of Other Gpcrs By Mutamentioning

confidence: 99%

“…We then used a PAFR antagonist or agonist as a pharmacological chaperone to analyze the structurally defective mutants after they were trafficked to the cell surface. Moreover, we used a new technique, site-specific Nterminal labeling of cell surface proteins on living cells by sortase-A (16,17), to evaluate the trafficking of mutant PAFR after stimulation with agonist. We found that a conserved proline, Pro 247 , in TM6 of PAFR is important not only for ER export but also for trafficking, e.g.…”

mentioning

confidence: 99%

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Hirota

Yasuda

Hashidate

et al. 2010

Journal of Biological Chemistry

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Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro 247 , in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

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“…Sortases are transpeptidase enzymes found in most Gram-positive bacteria that are specialized in this task. Among the different bacterial sortases isolated so far, the Staphylococcus aureus sortase A (SrtA) has been by far the most widely used for protein engineering (36). SrtA recognizes peptide substrates containing the sequence LPXTG (where X can be any residue), cleaving the peptide bond between the Thr and Gly residues.…”

Section: Backbone Cyclization Using Protease-catalyzed Transpeptidationmentioning

confidence: 99%

Biological Synthesis of Circular Polypeptides

Aboye

Camarero

2012

Journal of Biological Chemistry

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Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.Protein engineering is usually defined as the modification of the primary sequence of a protein to change in a defined way its three-dimensional structure and biological function. Site-specific mutagenesis using either recombinant DNA technology or chemical synthesis has been successfully applied to numerous proteins. Another useful approach to protein engineering involves changing backbone topology. Backbone cyclization or circularization (i.e. the covalent linkage of the N-terminal amino and C-terminal carboxylic groups) is a powerful tool for the study and manipulation of protein structure and function. The introduction of a covalent bond between the N and C termini of a protein provides a more stable topological constraint than the use of disulfide bonds. Hence, in proteins whose N and C termini are close in the native structure (a surprisingly common feature in folded proteins, particularly in single-domain proteins (1)), backbone cyclization should stabilize the protein fold by reducing the backbone entropy associated with the unfolded state of a protein. Backbone cyclization also has the added benefit of making proteins more resistant to proteolytic degradation, in particular against exoproteases (2, 3), which should increase both the in vitro stability of industrial enzymes and the in vivo stability of proteins with interesting pharmacological properties. In addition, protein cyclization provides a valuable tool for exploring fundamental questions in protein folding, in particular how topology affects the kinetics of protein folding (4).Backbone peptide cyclization has also been widely used in bioorganic and medicinal chemistry to improve the biochemical and biophysical properties of flexible and labile peptides in the development of peptide-based drug candidates (2, 3). The cyclization of flexible linear peptides reduces their conformational freedom and creates constrained structural frameworks that often confer high receptor binding affinity, specificity, and enhanced stability (2, 3).Backbon...

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Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

Cited by 190 publications

References 96 publications

A general strategy for the evolution of bond-forming enzymes using yeast display

A general strategy for the evolution of bond-forming enzymes using yeast display

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Biological Synthesis of Circular Polypeptides

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