A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study.

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.

Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing ß-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli.

BACKGROUND: HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA. RESULT: In this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L⁻¹ (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L⁻¹ HIV-1 CA. A two-step purification strategy (chitin beads + IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay. CONCLUSIONS: By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies.