Subcloning, expression, and purification of active (VlsPLA2WT) and inactive (VlsPLA2∆CD) proteins were performed using the Protein Expertise Platform at Umeå University. Briefly, E. coli Origami2 (DE3) cells were used and protein expression was induced by the addition of 0.4 mM isopropyl β‐d ‐1‐thiogalactopyranoside. The target protein was purified using His‐tag purification resin (Roche) according to the manufacturer's instructions. For the phospholipase A2 enzymatic activity assay, the EnzChek Phospholipase A2 Activity Kit was used (Thermo Fisher Scientific) according to the manufacturer's instructions. In all assays, 10 μM of pure active (VlsPLA2WT) or inactive (VlsPLA2∆CD) protein was used.
Enzchek phospholipase a2 activity kit
Manufactured by Thermo Fisher Scientific
The EnzChek Phospholipase A2 Activity Kit is a fluorescence-based assay designed to measure the activity of phospholipase A2 (PLA2) enzymes. PLA2 enzymes catalyze the hydrolysis of the sn-2 ester bond of phospholipids, releasing fatty acids and lysophospholipids. The kit uses a non-fluorescent phospholipid substrate that becomes fluorescent upon PLA2-catalyzed hydrolysis, allowing for the quantitative determination of PLA2 activity.
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2 protocols using enzchek phospholipase a2 activity kit
1
Purification and Activity Assay of VlsPLA2 Proteins
2
Cloning, Expression, and Purification of VlsPLA2
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The cloning, expression and purification of the VlsPLA2 protein was done at the Protein Expertise Platform (PEP) at Umeå University. Briefly, Origami2 (DE3) cells were grown in LB media supplemented with 50 μg/mL kanamycin at 37°C with agitation. The culture was incubated until an OD600 ~ 0.6-0.7 was achieved. Protein expression was induced by the addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated overnight at 20°C. Cells were harvested by centrifugation 4000 x g for 20min. The target protein was purified using His-Tag Purification Resin (Roche) according to the manufacturer. The His-Trx-tag was cleaved off using Prescission3C protease. Finally, the protein was run on a Superdex200 (Cytiva), 20mM NaP 8.0, 200mM NaCl, 0.5mM b-me. For the phospholipase A2 enzymatic activity, the EnzChek Phospholipase A2 activity kit was used (ThermoFisher), according to manufacturer instructions. In all assays, 10μM of pure active (VlsPLA2 WT ) and inactive (VlsPLA2 H98A/D99A ) protein was used, previously constructed by Integrated DNA Technologies (idtDNA).
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