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A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin
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...
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Published in: | Bio-protocol 2021-09, Vol.11 (18), p.e4158-e4158 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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
Src2 tyrosine kinase induces phosphorylation of cortactin as a mechanism to control lamellipodia protrusion and filopodia formation in cultured
bag cell neurons ( He
, 2015 ; Ren
, 2019 ). In order to provide
evidence for Src2-mediated phosphorylation of cortactin, we developed a robust and cost-effective method for the efficient expression and purification of
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
, 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
. |
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ISSN: | 2331-8325 2331-8325 |
DOI: | 10.21769/BioProtoc.4158 |