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

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 elusiv...

Full description

Saved in:
Bibliographic Details
Published in:Bio-protocol 2023-02, Vol.13 (3), p.e4608-e4608
Main Authors: Reichardt, Sven, Stintzi, Annick, Schaller, Andreas
Format: Article
Language:English
Subjects:
Biology
Methods
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by
cites
container_end_page e4608
container_issue 3
container_start_page e4608
container_title Bio-protocol
container_volume 13
creator Reichardt, Sven
Stintzi, Annick
Schaller, Andreas
description 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 ( ) 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.
doi_str_mv 10.21769/BioProtoc.4608
format article
fullrecord <record><control><sourceid>proquest_doaj_</sourceid><recordid>TN_cdi_doaj_primary_oai_doaj_org_article_52d423197f454ebc8659b7cc346cab0b</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><doaj_id>oai_doaj_org_article_52d423197f454ebc8659b7cc346cab0b</doaj_id><sourcerecordid>2779341631</sourcerecordid><originalsourceid>FETCH-LOGICAL-c413t-545aa4062b2ea6b8508179f421fbf66abbd8ee8ea98b2f74d8058449a327cc123</originalsourceid><addsrcrecordid>eNpVkU1P3DAQhqOqVUGUc29Vjr0E_BXHvlSiqxaQkFiJcnbHziRrlI1TO0Haf1-X3a7gZMt-_Mx43qL4TMkFo43Ul999WMcwB3chJFHvilPGOa0UZ_X7V_uT4jylJ0II5ZJRKT4WJ1wqKrUmp8Xvq5RgV3YhluvNboY0QcJqi62HGdtyjdPsWyzXEd0SU6ZWA8Iz9Fg-Jj_25cNunDc4e1feD74P04F_WGyaY1akT8WHDoaE54f1rHj8-ePX6qa6u7--XV3dVU5QPle1qAEEkcwyBGlVTRRtdCcY7WwnJVjbKkSFoJVlXSNaRWolhAbOGuco42fF7d7bBngyU_RbiDsTwJuXgxB7AzH3OaCpWSsYp7rpRC3QOiVrbbOFC-nAEptd3_auabF5FA7H_JfhjfTtzeg3pg_PJo9Uc0qy4OtBEMOfBdNstj45HAYYMSzJsKbRXFDJaUYv96iLIaWI3bEMJeYlZnOM2fyLOb_48rq7I_8_VP4X3WmneA</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2779341631</pqid></control><display><type>article</type><title>Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates</title><source>PubMed Central (PMC)</source><creator>Reichardt, Sven ; Stintzi, Annick ; Schaller, Andreas</creator><creatorcontrib>Reichardt, Sven ; Stintzi, Annick ; Schaller, Andreas</creatorcontrib><description>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 ( ) 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.</description><identifier>ISSN: 2331-8325</identifier><identifier>EISSN: 2331-8325</identifier><identifier>DOI: 10.21769/BioProtoc.4608</identifier><identifier>PMID: 36816990</identifier><language>eng</language><publisher>United States: Bio-Protocol</publisher><subject>Biology ; Methods</subject><ispartof>Bio-protocol, 2023-02, Vol.13 (3), p.e4608-e4608</ispartof><rights>Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC.</rights><rights>Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9909310/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9909310/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36816990$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Reichardt, Sven</creatorcontrib><creatorcontrib>Stintzi, Annick</creatorcontrib><creatorcontrib>Schaller, Andreas</creatorcontrib><title>Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates</title><title>Bio-protocol</title><addtitle>Bio Protoc</addtitle><description>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 ( ) 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.</description><subject>Biology</subject><subject>Methods</subject><issn>2331-8325</issn><issn>2331-8325</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>DOA</sourceid><recordid>eNpVkU1P3DAQhqOqVUGUc29Vjr0E_BXHvlSiqxaQkFiJcnbHziRrlI1TO0Haf1-X3a7gZMt-_Mx43qL4TMkFo43Ul999WMcwB3chJFHvilPGOa0UZ_X7V_uT4jylJ0II5ZJRKT4WJ1wqKrUmp8Xvq5RgV3YhluvNboY0QcJqi62HGdtyjdPsWyzXEd0SU6ZWA8Iz9Fg-Jj_25cNunDc4e1feD74P04F_WGyaY1akT8WHDoaE54f1rHj8-ePX6qa6u7--XV3dVU5QPle1qAEEkcwyBGlVTRRtdCcY7WwnJVjbKkSFoJVlXSNaRWolhAbOGuco42fF7d7bBngyU_RbiDsTwJuXgxB7AzH3OaCpWSsYp7rpRC3QOiVrbbOFC-nAEptd3_auabF5FA7H_JfhjfTtzeg3pg_PJo9Uc0qy4OtBEMOfBdNstj45HAYYMSzJsKbRXFDJaUYv96iLIaWI3bEMJeYlZnOM2fyLOb_48rq7I_8_VP4X3WmneA</recordid><startdate>20230205</startdate><enddate>20230205</enddate><creator>Reichardt, Sven</creator><creator>Stintzi, Annick</creator><creator>Schaller, Andreas</creator><general>Bio-Protocol</general><general>Bio-protocol LLC</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20230205</creationdate><title>Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates</title><author>Reichardt, Sven ; Stintzi, Annick ; Schaller, Andreas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c413t-545aa4062b2ea6b8508179f421fbf66abbd8ee8ea98b2f74d8058449a327cc123</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Biology</topic><topic>Methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Reichardt, Sven</creatorcontrib><creatorcontrib>Stintzi, Annick</creatorcontrib><creatorcontrib>Schaller, Andreas</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>Directory of Open Access Journals</collection><jtitle>Bio-protocol</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Reichardt, Sven</au><au>Stintzi, Annick</au><au>Schaller, Andreas</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates</atitle><jtitle>Bio-protocol</jtitle><addtitle>Bio Protoc</addtitle><date>2023-02-05</date><risdate>2023</risdate><volume>13</volume><issue>3</issue><spage>e4608</spage><epage>e4608</epage><pages>e4608-e4608</pages><issn>2331-8325</issn><eissn>2331-8325</eissn><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 ( ) 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.</abstract><cop>United States</cop><pub>Bio-Protocol</pub><pmid>36816990</pmid><doi>10.21769/BioProtoc.4608</doi><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 2331-8325
ispartof Bio-protocol, 2023-02, Vol.13 (3), p.e4608-e4608
issn 2331-8325
2331-8325
language eng
recordid cdi_doaj_primary_oai_doaj_org_article_52d423197f454ebc8659b7cc346cab0b
source PubMed Central (PMC)
subjects Biology
Methods
title Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-06T15%3A09%3A23IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_doaj_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Assay%20for%20Phytaspase-mediated%20Peptide%20Precursor%20Cleavage%20Using%20Synthetic%20Oligopeptide%20Substrates&rft.jtitle=Bio-protocol&rft.au=Reichardt,%20Sven&rft.date=2023-02-05&rft.volume=13&rft.issue=3&rft.spage=e4608&rft.epage=e4608&rft.pages=e4608-e4608&rft.issn=2331-8325&rft.eissn=2331-8325&rft_id=info:doi/10.21769/BioProtoc.4608&rft_dat=%3Cproquest_doaj_%3E2779341631%3C/proquest_doaj_%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c413t-545aa4062b2ea6b8508179f421fbf66abbd8ee8ea98b2f74d8058449a327cc123%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=2779341631&rft_id=info:pmid/36816990&rfr_iscdi=true