Phytaspase cleavage assay using synthetic oligopeptide substrates

Phytaspase cleavage assay using synthetic oligopeptide substrates
 

1. Expression of C-terminally His-tagged phytaspases in N. benthamiana 

Prior to the experiment, phytaspase expression cassettes are generated comprising the 35S promoter, the phytaspase cDNA with 6 terminal His codons and the OCS terminator. The expression cassettes are then cloned into a binary vector (pART27) and transformed into Agrobacterium tumefaciens C58C1. Agrobacteria containing an expression construct for the P19 suppressor of silencing are also needed. Agrobacteria carrying the empty expression vector are used as control. 

• Streak out several single colonies of A. tumefaciens C58C1 containing the phytaspase expression vector on LB plates with appropriate antibiotics (spectinomycin, rifampicin, tetracycline; rifampicin and tetracycline for the empty-vector control). Also streak out agrobacteria carrying the P19 expression construct using LB plates containing kanamycin instead of spectinomycin.
• Incubate for 48 hrs at 28 °C.
• Resuspend bacteria in 6 ml infiltration buffer (10 mM MES pH 5.6, 10 mM MgCl₂, 0.15 mM acetosyringone) and transfer the suspension to a 15 ml culture tube.
• Spin down at 1000 x g for 10 min, discard supernatant.
• Resuspend cells in 1 ml infiltration buffer, determine optical density (OD) at 600 nm.
• Mix the two suspensions carrying the phytaspase expression construct and the P19 silencing suppressor at an OD₆₀₀ ratio of 0.7 : 1.0 in 50 ml infiltration buffer.
• Use a syringe without needle to inject the suspension into the abaxial side of leaves of six-week old of N. benthamiana plants grown at 25°C and 16/8 h day/night cycle.
• Harvest the leaves after 5 days under the same growth regime.
• Remove the central vein. Place the remaining leaf material upper side down into a beaker containing approx. 100 ml extraction buffer (50 mM NaH₂PO₄/Na₂HPO₄, pH 6.5, 200 mM KCl) on ice.
• Vacuum infiltrate the extraction buffer in a desiccator at 75 mbar for 2 min, then slowly release the vacuum.
• Dry the leaves on blotting paper. Collect them into a syringe barrel plugged with glass wool.
• Place the syringe barrel into a centrifuge tube and spin for 7 min at 1500 x g and 4 °C.
• Transfer the apoplastic wash to a new tube and clear by centrifugation (20000 x g, 4°C).
• Transfer the supernatant into a new tube.
• Add imidazole to 4 mM final concentration.

2. Purification of phytaspases

His-tagged phytaspases are purified from apoplastic washes by metal chelate affinity chromatography on Ni-NTA agarose (Qiagen; Hilden, Germany) in a batch procedure. As a control, apoplastic washes from empty vector-infiltrated plants are subjected to the same purification procedure. Purified fractions are used for cleavage assays with synthetic oligopeptide substrates. For applications that require highest purity, for example the PICS (Proteomics Identification of Cleavage Sites) assay for the analysis of substrate selectivity, further purification by gel filtration is advised.

• Pipet 500 µl Ni-NTA agarose (50 % slurry) into a 15 ml culture tube and spin for 2 min at 500 x g to sediment the matrix
• Wash the matrix 3 times with 10 ml binding buffer (50 mM Na₂HPO₄/NaH₂PO₄ pH 7.0, 200 mM KCl, 4 mM imidazole), resuspend in 1 ml binding buffer after the final wash.
• Add the apoplastic wash from above and incubate for 1 h at 4°C on a rotating wheel.
• Collect the matrix by centrifugation as above, and wash 3 times with 12 ml binding buffer.
• Carefully remove the buffer after the last washing step.
• Add 600 µl elution buffer (50 mM Na₂HPO₄/NaH₂PO₄ pH 7.0, 200 mM KCl, 200 mM imidazole), sediment by centrifugation, recover the supernatant. Repeat twice.
• Combine the eluates and reduce the volume to about 100 µl by ultrafiltration using a Vivaspin centrifugal concentrator (30 k molecular weight cut-off; Sartorius Stedim; Stonehouse, UK).
• For buffer exchange, add 500 µl elution buffer without imidazole. Reduce the volume to 100 µl. Repeat three times.
• Add glycerol to 50 % final concentration, and store at -20 °C.
• Determine protein concentration using a commercial Bradford assay system.
• Assess purity by SDS-PAGE and Coomassie-Brilliant Blue staining.

For applications that require highest purity, further purification by gel filtration on a Superdex 200 HR 10/30 gel filtration column (GE Healthcare; Freiburg, Germany) is recommended. 

• Equilibrate the column on an on a ÄKTA purifier 900 chromatography system (GE Healthcare; Munich, Germany) in 50 mM sodium phosphate buffer pH 7.0, 300 mM NaCl, at 0.5 ml/min.
• Inject the eluate from the Ni-NTA matrix, after ultrafiltration for volume reduction to 100 µl, but before the addition of glycerol.
• Monitor UV absorbance at 280 and 254 nm; collect the column eluate in 200 µl fractions.
• Use the fraction at the peak maximum for further analyses. Alternatively, pool all peak fractions, concentrate by ultrafiltration as above, add glycerol to 50 % final concentration, and store at -20 °C.
• Determine protein concentration using a commercial Bradford assay system.
• Assess purity by SDS-PAGE and Coomassie-Brilliant Blue staining.

3. Peptide digest

Activity and cleavage specificity of the purified phytaspases is tested with synthetic oligopeptides as substrates. Lyophilized custom-synthesized synthetic peptides can be obtained at >90 % purity from commercial suppliers. In Reichardt et al., 2020 (doi: 10.1126/science.aaz5641) we used phytosufokine (PSK) peptide, with 5 additional amino acids at the N-terminus (EAHLD[sY]I[sY]TQ; N-terminal extension underlined), custom-synthesized by PepMic (Suzhou, China). 

• Resuspend the lyophilized peptide in ddH₂O; determine the concentration spectrophoto­metrically at 260 nm, based on the molar extinction coefficient for two sulfo-tyrosine residues (e₂₆₀ = 566 M^-1 cm^-1).
• Set up the in-vitro digest in 100 µl reaction buffer (50 mM NaH₂PO₄/Na₂HPO₄, pH 6.5, 200 mM KCl) containing 140 nM of the purified phytaspase, or an equal volume of the mock-purified fraction. Start the reaction by addition of 5 µM substrate peptide.
• The reaction is stopped by acidification with TFA. Since the activity of the protease of interest with the particular substrate peptide is usually not known, we recommend to take aliquots at different time points. For example, at 0 min, 10 min, 30 min, 1 h, 5 h, 24 h after addition of substrate remove 10 µl aliquots and add to 90 µl 0.1% TFA.

For subsequent identification and quantification of cleavage products by nanoLC-ESI-MS/MS, samples need to be desalted and concentrated on C18 ZIP tips or StageTips. The eluate is evaporated to dryness and resuspended in 20 µl 0.1 % (v/v) formic acid. One tenth of the sample is sufficient for MS analysis. 

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