A co-purification method for efficient production and Src kinase-mediated phosphorylation of aplysia cortactin

Sherlene L. Brown, Yuan Ren, Daniel M. Suter, Seema Mattoo

Research output: Contribution to journalArticlepeer-review

Abstract

Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent reports show that Aplysia Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured Aplysia bag cell neurons (He et al., 2015; Ren et al., 2019). In order to provide in vitro evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of Aplysia cortactin and Src2 kinase that can be used for biochemical studies including phosphorylation assays. By co-purifying cortactin and Src kinase with a phosphatase (YopH) from Yersinia enterocolitica, we eliminated the problem of non-specific phosphorylation of induced proteins by bacterial kinases and also reduced costs by bypassing the need for commercial enzymatic treatments. This protocol is reproducible and can be modified to produce homogenous non-phosphorylated proteins during recombinant protein expression in Escherichia coli.

Original languageEnglish (US)
Article numbere4158
JournalBio-protocol
Volume11
Issue number18
DOIs
StatePublished - Sep 20 2021
Externally publishedYes

Keywords

  • Aplysia
  • Cortactin
  • Growth cones
  • Neurons
  • Phosphorylation
  • Src
  • Tyrosine phosphatase
  • YopH

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology
  • Plant Science

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