Interferon-inducible protein 16 (IFI16) is a multifunctional p200-family protein that plays pivotal roles in p53-mediated apoptosis, tumor suppression and DNA damage response. Recently, another function of IFI16 in innate immune sensing and response has been uncovered, in which IFI16 recognizes the exogenous DNAs through cooperative binding of DNAs via its two DNA binding domains, HINa and HINb. Although the mechanism by which the HINb domain recognizes DNAs has been elucidated, the molecular basis of the cooperativity between HINa and HINb during DNA recognition process is still not clear. Here we report expression and purification of a truncated human IFI16 protein (HINab-GS) containing HINa in tandem with HINb with the joining region between HINa and HINb replaced by a short GS linker in Escherichia coli. DNA binding activities of HINab-GS to various double-stranded DNAs (dsDNAs) of different lengths were then examined using electrophoretic mobility shift assays. HINab-GS exhibited efficient binding activity to dsDNAs, and its DNA binding affinity correlated positively with the length of dsDNAs. A co-crystallization condition of HINab-GS bound to a 30 bp dsDNA derived from vaccinia virus was also found. Together, our work provides a basis for structurally elucidating the mechanism governing cooperative DNA recognition by IFI16. (C) 2014 Elsevier Inc. All rights reserved.