Flow‐Based Enzymatic Ligation by Sortase A

Policarpo, Rocco L.; Kang, Hansol; Liao, Xiaoli; Rabideau, Amy E.; Simon, Mark D.; Pentelute, Bradley L.

doi:10.1002/ange.201403582

Cited by 21 publications

(9 citation statements)

References 83 publications

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“…The high substrate specificity for such a short recognition sequence makes SrtA a versatile tool to modify proteins with a functional protein or a chemically modified peptide [16]. Indeed, various applications of SrtA have been reported, including protein ligation [17], cell‐surface protein labeling [18, 19], covalent protein immobilization on solid support [20], protein‐lipid conjugation [21], protein‐liposome conjugation [22], protein glycosylation [23], and peptide and protein cyclization [24, 25]. However, it is challenging to use SrtA for protein modification in living cells because its catalytic activity is strongly dependent on the Ca 2+ concentration [26], which is quite low (–100 nM) in living cells [27, 28] except for in the endoplasmic reticulum [29].…”

Section: Introductionmentioning

confidence: 99%

Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Hirakawa

Ishikawa

Nagamune

2015

Biotechnology Journal

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A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca(2+) concentrations and in the presence of Ca(2+)-binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+)-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations.

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Section: Introductionmentioning

confidence: 99%

Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Hirakawa

Ishikawa

Nagamune

2015

Biotechnology Journal

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“…Affinity immobilization strategies or flow-based platforms have also been used for the selective removal of reaction components (Policarpo et al . 2014; Warden-Rothman et al . 2013).…”

Section: Molecular Engineering Toolbox For Complex Biological Samplesmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

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Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

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“…For example, the Beck-Sickinger group immobilized SrtA onto an acrylamide-PEG co-polymer (PEGA) resin through Cu(I) catalyzed “click chemistry” to generate an immobilized biocatalyst with low enzymatic activity 50 . Pentelue and coworkers developed flow-based SML, in which SrtA was immobilized onto Ni-NTA agarose resin using a His-tag 16 . This protocol was particularly useful for preparing protein conjugates inaccessible by traditional solution batch ligation when low nucleophile concentrations were used.…”

Section: Introductionmentioning

confidence: 99%

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

Zhao

Hong

Cheng

et al. 2017

Sci Rep

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Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.

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Flow‐Based Enzymatic Ligation by Sortase A

Cited by 21 publications

References 83 publications

Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

A molecular engineering toolbox for the structural biologist

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

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Flow‐Based Enzymatic Ligation by Sortase A

Cited by 21 publications

References 83 publications

Ca2+‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Ca2+‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

A molecular engineering toolbox for the structural biologist

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

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Product

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Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli

Ca²⁺‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli