Native protein purification of ferroxidase LPR1 from leaf extracts of a transgenic Arabidopsis thaliana line

STAR Protoc. 2022 Dec 16;3(4):101733. doi: 10.1016/j.xpro.2022.101733. Epub 2022 Sep 29.

Abstract

LPR1 (LOW PHOSPHATE ROOT 1), a bacterial-type plant ferroxidase, is crucial for local root phosphate (Pi) sensing. Here, we present a detailed protocol for native (tag-free) protein purification of LPR1 from leaf extracts by differential ammonium sulfate precipitation, size exclusion, and cation exchange chromatography of a transgenic Arabidopsis thaliana line overexpressing LPR1. We outline steps for LPR1 purification tracking via immune blot analysis and ferroxidase activity assay. The protocol yields highly pure and active LPR1 protein for biochemical analysis. For complete details on the use and execution of this protocol, please refer to Naumann et al. (2022).

Keywords: Plant sciences; Protein biochemistry; Protein expression and purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis Proteins* / metabolism
  • Arabidopsis* / metabolism
  • Ceruloplasmin / metabolism
  • Oxidoreductases / metabolism
  • Phosphates / metabolism
  • Plant Roots / metabolism

Substances

  • Arabidopsis Proteins
  • Ceruloplasmin
  • Phosphates
  • LPR1 protein, Arabidopsis
  • Oxidoreductases