Expression, purification and kinetic characterisation of human tissue transglutaminase

Roy, Isabelle; Smith, Olivia Eilers; Clouthier, Christopher M.; Keillor, Jeffrey W.

doi:10.1016/j.pep.2012.10.002

Cited by 19 publications

(28 citation statements)

References 43 publications

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“…Trends are similar to those for gpTGase 2, and compound 5 b turned out to be the acyl donor with the most favourable substrate properties towards the human enzyme ( k cat / K m =78 900 m −1 s −1 ). A Michaelis constant similar to that of 5 b has been reported for the chromogenic analogue Z‐Glu(O p Np)‐Gly‐OH towards hTGase 2, with a similar tendency being found on comparison with the guinea pig enzyme …”

Section: Resultssupporting

confidence: 72%

“…AM ichaelis constant similar to that of 5b has been reportedf or the chromogenica nalogueZ -Glu(OpNp)-Gly-OHt owards hTGase 2, with as imilar tendency being found on comparison with the guinea pig enzyme. [31] An experimental setup with 96-well plates was used to record spontaneous and enzymatic hydrolyses simultaneously, so there is at ime delay between the start of the reaction and the data acquisition. In consequence, those substrate concentrationsp ipetted at earlier time points might be lower than intendeda tt he start of the measurement.…”

Section: Kinetic Analysis Of Tgase 2-catalysed Conversions Of the Glumentioning

confidence: 99%

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Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

et al. 2016

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Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

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Section: Resultssupporting

confidence: 72%

Section: Kinetic Analysis Of Tgase 2-catalysed Conversions Of the Glumentioning

confidence: 99%

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

et al. 2016

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“…Using these methods, the production of TG2 in a hexa-histidine labeled form has become routine [22,24,25], although other forms of TG2 can remain a challenge to obtain in good yield. A complementary technique for the purification of hTG2 was recently reported, in which hTG2 was expressed as a fusion with glutathione S-transferase (GST) and followed by a one-step affinity chromatography purification [26]. Unlike TG2, the purification of the most widely used MTG (from S. mobaraensis and homologs) is complicated by the fact that the native enzyme is expressed as a zymogen (pro-MTG); a 46-residue N -terminal pro-sequence must be proteolytically cleaved in order for MTG to be rendered functional.…”

Section: Production and Engineering Of Tgasesmentioning

confidence: 99%

Biotechnological Applications of Transglutaminases

Rachel

Pelletier

2013

Biomolecules

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In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. This specific function has long been exploited in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool, and leather. In recent years, biotechnological applications of transglutaminases have come to light in areas ranging from material sciences to medicine. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as many of their characteristics that remain poorly understood. Those studies also work towards the goal of developing transglutaminases as more efficient catalysts. Progress in this area includes structural information and novel chemical and biological assays. Here, we review recent achievements in this area in order to illustrate the versatility of transglutaminases.

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“…However, mutations may occur in the vector at non-desired sites during PCR amplification, which can compromise the fidelity of the approach. On the other hand, because the overlap extension and megaprimer methods utilize vectors that have been digested with restriction endonucleases, introduction of mutations in the vector region is avoided [ 24 – 26 ]. However, vector construction by these methods requires two sequential PCR reactions, and the purification of insert DNA fragments.…”

Section: Introductionmentioning

confidence: 99%

A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis

2015

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BackgroundSeamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥15 base pairs).ResultsTo extend the versatility of this system, I examined whether, in addition to bacterial extracts from the PPY strain, other E. coli laboratory strains were suitable for the SLiCE protocol. Indeed, carefully prepared cell extracts from several strains exhibited sufficient cloning activity for seamless gene incorporation into vectors with short homology lengths (approximately 15–20 bp). Furthermore, SLiCE was applied to the polymerase chain reaction (PCR)-based site-directed mutagenesis method, in a process termed “SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis)”. SLiP site-directed mutagenesis simplifies the steps of PCR-based site-directed mutagenesis, as it exploits the capability of the SLiCE method to insert multiple fragments.ConclusionsSLiCE can be performed in the laboratory with no requirement for a special E. coli strain, and the technique is easily established. This method increases the cloning efficiency, shortens the time for DNA manipulation, and greatly reduces the cost of seamless DNA cloning.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0162-8) contains supplementary material, which is available to authorized users.

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Expression, purification and kinetic characterisation of human tissue transglutaminase

Cited by 19 publications

References 43 publications

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

Biotechnological Applications of Transglutaminases

A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis

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