Expression and purification of functional insulin and insulin-like growth factor 1 holoreceptors from mammalian cells

Content Provider	Scilit
Author	Bovi, Richard J. Delle Miller, W. Todd
Copyright Year	2017
Description	Journal: Analytical biochemistry The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization. We screened a panel of 24 structurally diverse detergents for optimal ligand activation. The receptors purified in n-dodecyl-β-D-maltoside showed ligand-stimulated autophosphorylation and kinase activity, suggesting an intact transmembrane signaling mechanism. This convenient purification protocol can be used to produce high quantities of IR, IGF1R, or other RTKs, and can be adapted for other challenging membrane proteins.
Related Links	http://europepmc.org/articles/pmc5701837?pdf=render https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701837/pdf
Ending Page	77
Page Count	9
Starting Page	69
ISSN	00032697
e-ISSN	10960309
DOI	10.1016/j.ab.2017.08.011
Journal	Analytical biochemistry
Volume Number	536
Language	English
Publisher	Elsevier BV
Publisher Date	2017-11-01
Access Restriction	Open
Subject Keyword	Journal: Analytical biochemistry Biochemical Research Analytical Chemistry Insulin Receptor Insulin-like Growth Factor 1 Receptor Membrane Proteins Receptor Tyrosine Kinases Streptavidin-binding Peptide
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology Biophysics