Expression and purification of soluble recombinant CCR5-T4L protein in an <i>E</i>. <i>coli</i> system.

<p>A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in <i>E</i>. <i>coli</i>. The pET-20b expression vector was transformed into Rosetta 2 (DE3) golden BL21 pLysS cells and analyzed using Coomassie brilliant blue R-250. Lane M: protein marker; lane 1: uninduced bacterial lysate; lane 2: IPTG-induced bacteria lysate; lane 3: small amount of soluble fraction purified on a Ni-nitrilotriacetic acid (NTA) histidine-binding column; lane 4: small amount of membrane fraction purified using a Ni-nitrilotriacetic acid (NTA) histidine-binding column; lane 5: large amount of soluble fraction purified by fast protein liquid chromatography (FPLC) using an AKTA purifier; lane 6: large amount of membrane fraction purified by fast protein liquid chromatography (FPLC) using an AKTA purifier. B and C) Western blot analyses using the anti-6×His tag monoclonal or anti-human CCR5 monoclonal antibodies (3A9).</p

Recombinant CCR5-T4L down-regulates surface CCR5 expression in MDMs, and inhibits MDM migration and binding properties.

<p>A and B) Effect of soluble recombinant CCR5-T4L or CCL5 on surface CCR5 expression in MDMs. MDMs were treated with soluble recombinant CCR5-T4L (1 μg/ml), CCL5 (1 μg/ml), or PBS for 24 h at 37°C. Surface (A) and intracellular CCR5 (B) were analyzed using flow cytometry and the PE-conjugated monoclonal antibody (2D7). Recombinant CCL5 protein was used as a control. The cellular distribution of CCR5 receptors was analyzed by fixing and permeabilizing cells using BD Cytofix/Cytoperm buffer. Data shown are from one representative experiment that was independently repeated at least three times. C) Dose-dependent effects of CCR5-T4L or CCL5 on surface CCR5 expression in MDM. D and E) Dose-dependent effects on MDM migration by CCR5-T4L (D) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (E). F and G) Dose-dependent effects on [125I]-CCR5 binding by CCR5-T4L (F) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (G). H and I) Dose-dependent effects on CCL5-induced [³⁵S]GTPγS binding to membranes from human macrophages cells treated with CCR5-T4L (H) or CCL5 (100 nM) plus different concentrations of CCR5-T4L protein (I). Data are the mean ± SD of triplicate cultures, which are representative of three experiments.</p

Soluble recombinant CCR5-T4L suppresses HIV infection in macrophages.

<p>A and B) Effect of soluble recombinant CCR5-T4L or CCL5 on macrophages using cell-cell fusion assays (A) and single round virus infection assays (B). Macrophages cultured for 7 days were stimulated for 24 h at 37°C with CCR5-T4L (1 μg/ml) or CCL5 (1 μg/ml) prior to HIV-1 Env-mediated cell-cell fusion or single round virus infection. C, D, G, H, and I) Effect of soluble recombinant CCR5-T4L on HIV BaL infection in macrophages. Macrophages cultured for 7 days were stimulated with CCR5-T4L (1 μg/ml) for 24 h at 37°C prior to HIV-1 Bal infection. Cultured supernatant was collected 8 days post-infection, and cells were collected 12 days post-infection. Supernatants were subjected to RT assays (C), total RNA was evaluated for HIV-1 <i>gag</i> expression using real-time PCR (D), and total protein extracted from cells was evaluated for HIV-1 p24 protein expression by western blot analyses (G and H) and GAPDH (I). Representative blots from three independent experiments are shown. Densitometry analyses of the blot were performed using Image J 1.44 software (NIH) and plotted into graphs (n = 3). E and F) CCR5-T4L suppresses HIV-1 replication in macrophages. Macrophages were cultured at 37°C for 24 h in conditioned media in the presence or absence of CCR5-T4L (1 μg/ml) prior to HIV-1 infection or simultaneously or 8 h post-infection. Supernatants were collected 8 days post-infection, and cells were collected 12 days post-infection. Supernatants were subjected to RT assays (E) and total RNA was evaluated for HIV-1 gag expression using real-time PCR (F). Data are expressed as RNA levels relative to control. The results represent the mean ± SD of three independent experiments.</p