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The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

Jin Q, Chen H, Wang X, Zhao L, Xu Q, Wang H, Li G, Yang X, Ma H, Wu H, Ji X - PLoS ONE (2015)

Bottom Line: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization.We also explored the possible mechanisms underlying the observed antiviral effects.This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu Province, China; Department of Neurology, The People's Hospital of Jiangsu Province, 300 Guangzhou Road, Nanjing, Jiangsu Province, China; Department of Neurology, Nanjing First Hospital, 68 Changle Road, Nanjing, Jiangsu Province, China.

ABSTRACT

Background: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.

Methods: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.

Results: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.

Conclusions: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

No MeSH data available.


Related in: MedlinePlus

Expression and purification of soluble recombinant CCR5-T4L protein in an E. coli system.A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in E. coli. 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).
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pone.0131894.g002: Expression and purification of soluble recombinant CCR5-T4L protein in an E. coli system.A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in E. coli. 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).

Mentions: Large-scale expression was successfully performed in a 5 liter shaking flask filled with 1 liter LB culture media. The expression of recombinant CCR5-T4L protein was induced by IPTG, and recombinant CCR5-T4L was induced with a final concentration 1 mM IPTG at 20°C for 48 h. Cell pellets were stable at -80°C for at least six months. In order to optimize high-level CCR5-T4L production, many variables were systematically studied, including timing, length of induction, and incubation temperature. The yield was significantly higher at 20°C than at 37°C. A further decrease of temperature to 15°C or a decrease of IPTG concentration from 1 mM to 0.4 mM resulted in lower yields. In addition, increasing cell density successfully maximized the yield. The highest recombinant protein expression was observed 48 h post induction at OD600 around 2.6. E. coli cell lysates were separated into the soluble cytoplasmic fraction (sol) and insoluble membrane fraction (mem). After a two-step purification from the soluble cytoplasmic fraction sample, we obtained soluble recombinant CCR5-T4L protein with a single visible band (~95% pure as estimated by SDS-PAGE). Soluble recombinant CCR5-T4L protein was found in both soluble and membrane fractions at the same molecular weight of approximately 85 kDa (Fig 2A). Western blot analyses with the anti-human CCR5 antibody (NT) and anti-His tag antibody (anti-6x his) confirmed the identity of the 85 kDa band (Fig 2B and 2C).


The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

Jin Q, Chen H, Wang X, Zhao L, Xu Q, Wang H, Li G, Yang X, Ma H, Wu H, Ji X - PLoS ONE (2015)

Expression and purification of soluble recombinant CCR5-T4L protein in an E. coli system.A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in E. coli. 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).
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pone.0131894.g002: Expression and purification of soluble recombinant CCR5-T4L protein in an E. coli system.A) Large scale purification and identification of recombinant CCR5-T4L. Recombinant CCR5-T4L was expressed in E. coli. 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).
Mentions: Large-scale expression was successfully performed in a 5 liter shaking flask filled with 1 liter LB culture media. The expression of recombinant CCR5-T4L protein was induced by IPTG, and recombinant CCR5-T4L was induced with a final concentration 1 mM IPTG at 20°C for 48 h. Cell pellets were stable at -80°C for at least six months. In order to optimize high-level CCR5-T4L production, many variables were systematically studied, including timing, length of induction, and incubation temperature. The yield was significantly higher at 20°C than at 37°C. A further decrease of temperature to 15°C or a decrease of IPTG concentration from 1 mM to 0.4 mM resulted in lower yields. In addition, increasing cell density successfully maximized the yield. The highest recombinant protein expression was observed 48 h post induction at OD600 around 2.6. E. coli cell lysates were separated into the soluble cytoplasmic fraction (sol) and insoluble membrane fraction (mem). After a two-step purification from the soluble cytoplasmic fraction sample, we obtained soluble recombinant CCR5-T4L protein with a single visible band (~95% pure as estimated by SDS-PAGE). Soluble recombinant CCR5-T4L protein was found in both soluble and membrane fractions at the same molecular weight of approximately 85 kDa (Fig 2A). Western blot analyses with the anti-human CCR5 antibody (NT) and anti-His tag antibody (anti-6x his) confirmed the identity of the 85 kDa band (Fig 2B and 2C).

Bottom Line: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization.We also explored the possible mechanisms underlying the observed antiviral effects.This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu Province, China; Department of Neurology, The People's Hospital of Jiangsu Province, 300 Guangzhou Road, Nanjing, Jiangsu Province, China; Department of Neurology, Nanjing First Hospital, 68 Changle Road, Nanjing, Jiangsu Province, China.

ABSTRACT

Background: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.

Methods: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.

Results: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.

Conclusions: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

No MeSH data available.


Related in: MedlinePlus