Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

Zhulenkovs, Dmitrijs; Jaudzems, Kristaps; Zajakina, Anna; Leonchiks, Ainars

doi:10.1016/j.bej.2013.11.018

Cited by 18 publications

(28 citation statements)

References 34 publications

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“…CyM6 retained 99 % of its specific activity after cyclization when compared to the linear SaSrtA M6 (Figure S8). This result is in agreement with a previous report, showing that SaSrtA WT retained full activity after cyclization [32] …”

Section: Methodssupporting

confidence: 94%

“…Regarding stabilities, CyM6 showed 4.6 and 2.6‐fold resistance (vs. rM4) in 1 m GdnHCl and 2.5 m urea, respectively (Figure 4 b). The obtained resistance of CyM6 is in good agreement with previous studies in which enhanced resistance (4.5‐fold) of cyclized SaSrtA WT in GdnHCl and urea was observed [31, 32] …”

Section: Methodssupporting

confidence: 91%

“…green fluorescent protein, [45] cytokines [46] ) towards thermal incubation, protease digestion, or chaotropic agents [43] . HtTBC of SaSrtA WT was previously implemented for enhanced stability against urea using an intein‐mediated posttranslational modification [32] . To further improve stability properties, HtTBC of SaSrtA M6 was performed using a sortase A from Streptococcus pyogenes (SpSrtA).…”

Section: Methodsmentioning

confidence: 99%

“…A few studies reported the engineering of SaSrtA WT for increased stability towards thermal and chemical denaturation (e.g. 11.2 °C improvement in melting temperature (T m ) and 4.5‐fold resistance in presence of 2.5 m urea) [31, 32] . However, engineering of highly active SaSrtA variants with enhanced tolerance against thermal and chemical stress has not been reported yet.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Zhi

Maté

Nöth

et al. 2020

Chemistry A European J

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Staphylococcus aureus sortase A (SaSrtA) is widely used for site‐specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head‐to‐tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6‐fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6‐fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

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Section: Methodssupporting

confidence: 94%

Section: Methodssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Zhi

Maté

Nöth

et al. 2020

Chemistry A European J

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“…The total fluorescence was recorded at 1-min intervals for 1 h, and the progress curves were constructed. The initial velocities of the biphasic reactions were obtained through nonlinear regression, as previously described [40,41]. The IC 50 values were determined by fitting the obtained data to the following default four-parameter variable slope sigmoidal function in SigmaPlot 12.5 using a nonlinear least squares algorithm: y=min+(max−min)1+(truexIC50)−HillSlope…”

Section: Methodsmentioning

confidence: 99%

Discovery of a New Class of Sortase A Transpeptidase Inhibitors to Tackle Gram-Positive Pathogens: 2-(2-Phenylhydrazinylidene)alkanoic Acids and Related Derivatives

et al. 2016

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A FRET-based random screening assay was used to generate hit compounds as sortase A inhibitors that allowed us to identify ethyl 3-oxo-2-(2-phenylhydrazinylidene)butanoate as an example of a new class of sortase A inhibitors. Other analogues were generated by changing the ethoxycarbonyl function for a carboxy, cyano or amide group, or introducing substituents in the phenyl ring of the ester and acid derivatives. The most active derivative found was 3-oxo-2-(2-(3,4dichlorophenyl)hydrazinylidene)butanoic acid (2b), showing an IC 50 value of 50 µM. For a preliminary assessment of their antivirulence properties the new derivatives were tested for their antibiofilm activity. The most active compound resulted 2a, which showed inhibition of about 60% against S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538 and S. epidermidis RP62A at a screening concentration of 100 µM.

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Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation

Zou,

Ji,

Schwaneberg

2023

Angewandte Chemie

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Sortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

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Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

Cited by 18 publications

References 34 publications

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Discovery of a New Class of Sortase A Transpeptidase Inhibitors to Tackle Gram-Positive Pathogens: 2-(2-Phenylhydrazinylidene)alkanoic Acids and Related Derivatives

Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation

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