ALLINI BI/D, LEDGIN-6, ALLINI-2, and KF116 were synthesized as previously described.25 (link),29 (link),31 (link),40 (link) WT HIV-1 IN and LEDGF/p75 recombinant proteins with 6 × His or FLAG tags were expressed in E. coli cells and purified as described.31 (link) Expression and purification of the complex between His-LEDGF/p75 and FLAG-HIV-1 IN was performed as follows. His-LEDGF/p75 was expressed in E. coli from pFT-1-LEDGF derived from pRSETB (Invitrogen).31 (link) FLAG-HIV-1 IN was expressed in E. coli as previously reported.31 (link) Cell pellets of both were mixed and lysed by sonication in buffer containing 500 mM NaCl, 50 mM HEPES at pH 7.5, 2 mM β-mercaptoethanol, 20 mM Imidazole, and 1 × Complete protease inhibitor (Roche). After centrifugation, the soluble lysate was filtered and purified using a 5 mL HisTrap (GE Healthcare) chromatography column. Bound protein was eluted using a linear gradient of 20 mM to 500 mM imidazole in the same buffer. Fractions containing both proteins were pooled and concentrated using a 100 kDa MWCO spin column. The proteins were then subjected to size exclusion chromatography using a HiLoad 16/60 Superdex 200 (GE Heathcare) in 500 mM NaCl, 50 mM HEPES at pH 7.5, and 2 mM β-mercaptoethanol. Fractions that contained the complex between His-LEDGF/p75 and FLAG-HIV-1 IN were pooled and concentrated using a 100 kDa MWCO spin column.