Abstract:Chromatin remodeling serves as a critical mechanism for gene regulation, enabling the replacement of canonical histones with their variants through the assistance of histone chaperones. Our study focused on the process by which the histone variant H2A.Z replaces H2A, mediated by the mammalian SRCAP chromatin remodeling complex subunit hYL1. Previous approaches typically involved separately expressing the two proteins and validating their interaction in vitro. In contrast, we designed an artificial single‐chain hH2B‐hH2A.Z (hTBZ) construct, co‐transformed the hYL1 gene with hTBZ, and co‐expressed the proteins in E. coli BL21(DE3). We then performed purification, fast protein liquid chromatography (FPLC), and SDS‐PAGE analysis. This approach successfully yielded the complexed proteins, confirming the in vivo interaction between hYL1 and hTBZ. © 2025 Wiley Periodicals LLC.Basic Protocol: Co‐transformation of hTBZ/YL1 into BL21 (DE3) competent cells, followed by induction and acquisition of protein