Computational protocol: Constitutive expression of active microbial transglutaminase in Escherichia coli and comparative characterization to a known variant

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Protocol publication

[…] Transformed E. coli were grown on LB plates containing kanamycin overnight at 37 °C and seeded into 500 ml Terrific Broth containing 25 μg/mL kanamycin. After incubation for 60 h at 25 °C, the samples were centrifuged at 4,000 rpm for 15 min and 4 °C. The resulting pellet was resuspended in binding buffer (500 mM NaCl, 30 mM imidazole) and thrice passed through a homogenizer (EmulsiFlex-C3, AVESTIN Inc.; Ottawa, Canada) at 1,500 bar. The cell debris was removed from the supernatant by centrifuging twice at 16,000 g for 20 min at 15 °C, whereas the supernatant was applied to a nickel affinity column (HisTrap 5 ml; GE Healthcare; Buckinghamshire, United Kingdom) primed with the binding buffer.Elution was performed using a linear gradient with an appropriate buffer (500 mM NaCl, 500 mM imidazole). The fractions containing TG were collected and dialyzed against a phosphate buffer at 4 °C (50 mM sodium phosphate buffer pH 8). Protein concentration was calculated with a NanoDrop 2000 (Thermo Fisher Scientific Inc. Massachusetts, USA) using a molar extinction coefficient of 71,850 1/M⋅cm and molecular weight of 39.07 kDa, calculated using the ProtParam (Swiss Institute of Bioinformatics) online software. [...] Kinetic constants were calculated using the coupled enzyme assay [], in which the ammonia release from mTG activity is coupled to the uptake by glutamate dehydrogenase (GDH). The ammonia, which is the byproduct of mTG catalyzed transamidation reaction between Z-Gln-Gly and hydroxylamine, was used as a substrate for GDH.The activity of GDH is dependent on NADH as a co-factor, whose disappearance can be monitored at 340 nm. 180 μL reaction mixture per microplate well contained 0.2 M MOPS pH 7.2, 1 mM EDTA, 10 mM α-KG, 2U GDH, 10 mM hydroxylamine, 0.5 mM NADH, and 0.25-50 mM Z-Gln-Gly final concentration. The plate was equilibrated for 5 min at 37 °C prior to addition of 20 μl mTG (1 U) or water instead of mTG as a blank. The concentration of NADH was calculated based on a calibration curve. Reactions were carried out in a 96 multi-well plate, and the absorbance was recorded in a BioTek EON plate reader (Vermont, USA). Kinetic constants were calculated using the Enzyme Kinetics toolbox in SigmaPlot 12 (Systat Software Inc.). […]

Pipeline specifications

Software tools ProtParam, SigmaPlot
Applications Miscellaneous, Protein physicochemical analysis
Organisms Escherichia coli
Chemicals Dimethyl Sulfoxide, Glutamic Acid, 2-Propanol