Virol

Virol. evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, by engineered lactobacillus has been proposed as one means to SJB3-019A improve the availability of HIV inhibitors. It is reasoned that, being a normal component of the vaginal flora, engineered lactobacilli will provide long-term stable expression of anti-HIV inhibitors. Engineered lactobacilli have been shown to inhibit HIV infection when tested (5, 6, 36); however, it is unclear that stable expression of inhibitors by the engineered lactobacilli will be maintained given the abundant and dynamic normal flora of the female genital tract. We propose to develop an alternative bacterial expression system SJB3-019A using a nonpathogenic bacterium that presents HIV-blocking agents efficiently, is inexpensive to produce as a killed, stabilized agent, and is likely to be compatible with exposure to human mucosal tissue. is a harmless soil and water bacterium that elaborates a protein surface layer (S layer) composed of a 98-kDa monomer protein (RsaA). It is secreted at high levels (20 to 25% of cell protein [17]) and self-assembles on the surface of the bacterium in a hexagonal pattern. A typical cell has approximately 40,000 copies of this protein monomer (25, 33, 34). We have demonstrated that it is feasible to insert large genetic segments within the S-layer gene while maintaining all functional aspects: SJB3-019A secretion, assembly SJB3-019A (crystallization), and cell surface attachment of the resulting recombinant protein, as well as consequent high density presentation of the inserted peptide (12, 17, 25, 26). S-layer expression/display of CD4 (domain 1) and MIP1 has successfully generated recombinant caulobacters that can inhibit HIV infection (27). Protein G from strains were grown at 30C in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCl2, 0.02% MgSO4) with 1.2% agar for plates. DH5 was used for cloning manipulations and was grown at 37C in Luria broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract) with 1.3% agar for plates. Ampicillin (Amp) and kanamycin (Km) were used at 50 g/ml, and chloramphenicol (Cm) was SJB3-019A used at 20 g/ml for and 2 g/ml for or was performed as previously described (13). The strains used were modifications of strain CB2A, which has a spontaneous amber mutation in (12). p4A containing a variant of with a BamHI site inserted at a position corresponding to amino acid 723 and a version further derived to contain domain 1 of CD4 have been described (27). Strain constructs maintaining these vectors will be referred to as C-C (control) and C-CD4, respectively. A construct displaying a version of protein G were constructed by gene replacement of the chromosomal gene of JS4022 with a modified gene containing a protein G construct (three GB1 IgG Fc binding domains, flanked and separated by 20 amino acid spacers) positioned at the amino acid 723 site (26). This was done by subcloning the displaying both protein G and CD4 was constructed by ligating the cells displaying protein G (C-PG) or protein G and CD4 (C-PG/CD4) were grown in peptone-yeast extract (PYE) medium overnight at 30C. After cells were washed by centrifugation and suspension Rabbit Polyclonal to Cyclosome 1 and the optical density at 600 nm was measured, the cell density was adjusted to 5 108 bacteria/ml in PYE. To capture antibody, 400 l of cells was combined with 100 l of human monoclonal antibody at 20 g/ml. Antibodies and specificities are listed in Table 1, and.