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. 2023 Feb 5;13(3):e4608.
doi: 10.21769/BioProtoc.4608.

Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates

Sven Reichardt  1 , Annick Stintzi  1 , Andreas Schaller  1
Affiliations

Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates

Sven Reichardt et al. Bio Protoc. .

Abstract

Proteases control plant growth and development by limited proteolysis of regulatory proteins at highly specific sites. This includes the processing of peptide hormone precursors to release the bioactive peptides as signaling molecules. The proteases involved in this process have long remained elusive. Confirmation of a candidate protease as a peptide precursor-processing enzyme requires the demonstration of protease-mediated precursor cleavage in vitro. In vitro cleavage assays rely on the availability of suitable substrates and the candidate protease with high purity. Here, we provide a protocol for the expression, purification, and characterization of tomato (Solanum lycopersicum) phytaspases as candidate proteases for the processing of the phytosulfokine precursor. We also show how synthetic oligopeptide substrates can be used to demonstrate site-specific precursor cleavage. Graphical abstract.

Keywords: Enzyme assay; Nicotiana benthamiana; Phytaspase; Protease; Protein purification; Substrate specificity; Synthetic peptide substrate; Transient expression.

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Conflict of interest statement

Competing interestsNo financial or non-financial competing interests are declared.

Figures

Video 1.
Video 1.. Agroinfiltration of N. benthamiana plants
Figure 1.
Figure 1.. Transient protein expression in N. benthamiana leaves.
A. Infiltration of agrobacterial suspension into the abaxial side of N. benthamiana leaves. B. Harvesting of leaves into extraction buffer. C. Leaves on blotting paper after vacuum infiltration. D, E. Leaves are piled one on top of the other and rolled up (D), in order to place them into the barrel of a 100 mL plastic syringe (E). (F) Diagram of a syringe barrel placed into a centrifuge bottle.
Figure 2.
Figure 2.. Purification of tomato phytaspases from apoplastic extracts of agroinfiltrated N. benthamiana plants.
A. SDS-PAGE analysis of phytaspases purified from apoplastic extracts by metal-chelate affinity chromatography on Ni-NTA agarose. Approximately 2 (Phyt4), 5 (Phyt5), or 6 µg (Phyt1, Phyt2, P69A) of protein were loaded onto the gel. A Coomassie-stained 10% gel is shown; the molecular mass of marker proteins is indicated in kDa. B. Further purification of Phyt2 by gel permeation chromatography. The elution volume is shown in milliliters. Protein elution was monitored at 280 nm and is shown as m(illi) A(bsorbance) U(nits) in blue; 200 µL fractions were collected and assayed for Phyt2 activity using a fluorogenic peptide substrate. Activity is shown in arbitrary units in red, as relative fluorescence increase per minute. Modified from Reichardt et al. (2018), Figures 3a and 4a (Reichardt et al., 2018).
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