Molecular cloning, overexpression, and an efficient one-step purification of α5β1 integrin

Abstract

The α5β1 integrin heterodimer is involved in many cellular processes and is an anticancer 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 α5β1 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 α5β1 integrin in quantities suitable for an array of different experiments including structural biology.

Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli

“…Regarding the current data, researchers have tried to overexpress integrin b1 within different eukaryotic expression systems, such as CHO cells (derived from Chinese hamster) [15], SF9 cells (isolated from Spodoptera frugiperda) [21], but hardly found any prokaryotic expression of it. There are several advantages of prokaryotic expression compared to a eukaryotic one, for example, shorter generation time, ease of handling and established fermentation technology with higher yield [22].…”