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Published on: December 5, 2016
Expression and Purification of the Type II Metacaspase from a Unicellular Green Alga Chlamydomonas reinhardtii
Igor Sabljić1, Yong Zou2, Marina Klemenčič3,4
1Uppsala BioCenter, Department of Molecular Science, Swedish University of Agricultural Sciences, Uppsala, Sweden. igor.sabljic@slu.se.
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.
Insights
Researchers developed a fast method to purify active Type II metacaspase (MCA) from the green alga Chlamydomonas reinhardtii. This protocol yields significant amounts of the crucial enzyme for further study.
Area of Science:
- Biochemistry
- Molecular Biology
- Protease research
Background:
- Type II metacaspases (MCAs) are proteases in the C14B MEROPS family.
- They cleave substrates after Arg or Lys residues.
- Previous studies successfully expressed and purified plant-derived Type II MCAs.
Purpose of the Study:
- To establish a protocol for the expression and purification of the sole Type II MCA from Chlamydomonas reinhardtii (CrMCA-II).
- To ensure the purified enzyme is proteolytically active.
Main Methods:
- Overexpression of CrMCA-II in E. coli.
- A two-step purification process: immobilized metal affinity chromatography (IMAC) with cobalt, followed by size-exclusion chromatography (SEC).
Main Results:
- A protocol yielding 4 mg of CrMCA-II protein per liter of culture was established.
- The purification process can be completed within one day.
- The purified CrMCA-II protein is proteolytically active.
Conclusions:
- A rapid and efficient method for purifying active Chlamydomonas reinhardtii Type II metacaspase has been developed.
- This protocol facilitates further research into the function and properties of CrMCA-II.
