Column-free purification of functional HIV-1 capsid protein and its application in assembly and inhibitor assays.

The HIV-1 capsid protein (CA) is essential for viral replication and serves as a validated antiviral drug target. Traditional purification of CA relies on multi-step chromatographic protocols, which are time-consuming and labor-intensive. In this study, we established a rapid, column-free purification strategy using a cleavable self-aggregating tag (cSAT) to produce functional wild-type CA protein from E. coli with >95 % purity within a single day. The workflow is compatible with high-throughput formats and scalable from microplates to fermenters, offering significant advantages over conventional purification methods. The purified CA retained full biological activity, as demonstrated by its ability to assemble into higher-order structures in a salt- and protein concentration-dependent manner in vitro. We further evaluated the effects of two well-characterized capsid modulators: CAI, a peptide inhibitor, and lenacapavir (LEN), a clinically approved capsid-targeting drug. Turbidity-based assembly assays confirmed that CAI inhibited and LEN enhanced CA assembly in a dose-dependent manner. When co-administered, CAI and LEN exhibited mutually antagonistic effects. Preincubation with CAI abolished LEN-mediated enhancement, indicating a potential conformational lock imposed by CAI. These findings demonstrate that the column-free strategy enables efficient production of functionally active CA protein suitable for downstream biochemical and inhibitor screening assays. The approach provides a practical tool for accelerating HIV-1 capsid research and antiviral discovery.