A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α

A class of extracellular stimuli requires activation of IKK1/alpha to induce generation of an NF-kappa B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-kappa B subunit, ReIB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/alpha primarily remains associated with IKK2/beta and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/a within the larger or the smaller complex pool. Constitutive activity of IKK1/alpha has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/alpha, and enable its use as a drug target, we expressed recombinant IKK1/alpha in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/alpha in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.