Purpose Integration of viral DNA into its host chromosome is an important step in the HIV replication process (7). HIV integrase is an important enzyme which plays an essential role in inserting the vital DNA into the host chromosome and replicating HIV (6). It is one of the three enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. Though combining antiviral therapy with protease and reverse transcriptase inhibitors has proved the improvement of antiviral therapy for treatment of AIDS. However, because there are still some problems for drug resistance and toxicity, we still need to do some additional researches on the antiviral drugs. Integrase is a good target for antivirals due to its necessity for HIV replication. In addition, integrase can only use a single active site to accommodate two different configurations of DNA substrates, which will constrain the process of HIV to develop drug resistance to integrase inhibitors (7).
To achieve the goal of crystallizing IN, our lab has been focusing much attention on the proteins that are binding partners of IN or host cofactors known to alter the biochemical characteristics of HIV-IN. One such host cofactor is SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 or also known as Ini1/SNF5. The goal of my research will be to express, purify, and characterize various Ini1 fragments as a potential chaperone in the crystallization of IN. And to find Ini1’s correct residues which binds to IN.
Methods We cloned three different Ini1 constructs into the Sumo-Pro expression vector. We used PCR to get three lengths of fragments and inserted our construct into the SUMO vector which encodes a SUMO expression chaperone as well as a 6-Hisitidine affinity tag for IMAC purification. We tested for expression of the
Sumo-Ini1 fragments through common methods utilizing the T7 based expression systems engineered into the Sumo-Pro vector. After small scale expression (5ml or 100ml cultures) and lysis via sonication, Ini1 was affinity purified with Ni-NTA and then by size-exclusion chromatography. Once the best behaved Ini1 construct was identified, we produced the protein in multiple 1L scale and purified to >90% purity. The SUMO tag includes an N-terminal recognition site that is recognized by the SUMO protease and will be removed. The purified protein then will be screened for crystal conditions.
Results Size exclusion chromatography showed us a single, homogeneous, peak when Ini1 protein fragment from residue 170 to residue 250 was dialyzed with IN. In addition, after purification, both IN and Ini1 was contained in this single peak. This confirmed that we had a Ini1 fragment that was selectively binding to IN and forming a complex upon mixing and dialyzing. The discovery of this stable complex is the first step in our attempts to stabilize IN for our crystallographic pursuits.