Abstract

Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.

Keywords

Metacaspase; Cobalt IMAC; Overexpression; Protease

Published in

Methods in Molecular Biology
2022, number: 2447, pages: 13-20
Title: Plant Proteases and Plant Cell Death : Methods and Protocols
ISBN: 978-1-0716-2078-6, eISBN: 978-1-0716-2079-3
Publisher: Humana

SLU Authors

• Sabljic, Igor

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Zou, Yong

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Ståhlberg, Jerry

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Bozhkov, Peter

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Biochemistry and Molecular Biology


Publication identifier

DOI: https://doi.org/10.1007/978-1-0716-2079-3_2

Permanent link to this page (URI)

https://res.slu.se/id/publ/120079