Mouse Aurora A: expression in Escherichia coli and purification.
Aurora kinases have recently become some of the most intensely pursued oncology targets for the design of small-molecule inhibitors. Most of the active Aurora-A protein variants are currently being expressed from baculoviruses in insect cells, while catalytically impaired proteins can also be generated in and purified from Escherichia coli. In this study we present a method of expressing large quantities of active mouse Aurora-A kinase domain as an N-terminal glutathione-S-transferase fusion protein in bacteria and outline a simple purification method that produces greater than 99% pure protein samples suitable for enzymatic assays and X-ray crystallography. The methods described in this report simplify mouse Aurora-A expression and purification, and may aid in the production of other difficult kinases in prokaryotes.
Elling
Robert A
RA
Department of Protein Sciences and Structural Biology, Sunesis Pharmaceuticals, Inc., 341 Oyster Point Blvd., South San Francisco, CA 94080, USA.
Tangonan
Bradley T
BT
Penny
David M
DM
Smith
Jeremy T
JT
Vincent
Diana E
DE
Hansen
Stig K
SK
O'Brien
Tom
T
Romanowski
Michael J
MJ
D000818:Animals; D064096:Aurora Kinase A; D064127:Aurora Kinases; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C576918:Aurka protein, mouse; D064096:Aurora Kinase A; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases