The alpha 5 beta 1 integrin heterodimer is involved in many cellular processes and is an anti-cancer therapeutic target. Therefore, access to quantities of protein suitable for studies aimed at understanding its biological functions is important. To this end, a large-scale protein expression system, utilizing the recombinant baculovirus/SF9 insect cell expression system, was created to produce the extracellular domain of the alpha 5 beta 1 integrin. An incorporated 8X-histidine tag enabled one-step nickel-column purification. Following sequence confirmation by LC-MS/MS, the conformation of the heterodimer was characterized by native dot blot and negative stain electron microscopy. Cellular transduction inhibition studies confirmed biological activity. The system allows expression and purification of alpha 5 beta 1 integrin in quantities suitable for an array of different experiments including structural biology. (C) 2013 Elsevier Inc. All rights reserved.