Microarray-based analysis of recombinant protein production in E.coli

The production of heterologous proteins in E. coli is a powerful tool in the generation of many important biotechnological and medical products. Despite its widespread use as an expression host, however, yields of correctly folded, functional protein are frequently low in E. coli. This is due largely to the formation of insoluble protein aggregates and to premature lysis of the bacterial cells. We, and others, have previously shown that the cell lysis phenomenon associated with recombinant protein production in E. coli is not a direct result of synthesis of heterologous proteins [1], [2]. Instead, protein production triggers a global stress response in the bacterium, but the mechanism by which cell lysis subsequently occurs remains unclear [3]

Cytokine secretion by DC:T-cell and B:T-cell co-cultures.

<p>(<b>A</b>) and (<b>B</b>): secretion of IL-2 (left) and IFN-γ (right) after 1 day co-culture of CD8+ OT-I T-cells with DCs or B-cells pre-pulsed or not with Ova peptide (10 pg–10 µg/ml). *p<0.01; **p<0.001, unpaired <i>t</i>-test. Bars indicate mean ± s.e.m. Data are representative of three independent experiments.</p

Differential Strength of Mechanical Forces between APC:T-cell Conjugates.

<p>(<b>A</b>) Schematic illustration of AFM experiments. A T-cell-mounted AFM cantilever was placed above a DC or B-cell that was firmly attached to a glass cover slip. The T-cell was then brought into contact with the target cell. Interaction forces were measured by the deflection of the cantilever after a pre-defined contact time. (<b>B</b>) Corresponding force-distance curve of DC:T-cell (black) or B:T-cell (red) interactions. F represents maximal interaction force (double arrow). (<b>C</b>) Interaction forces of DC:T-cell (black) or B:T-cell conjugates (red) in the absence (-Ova) or presence of Ova peptide (+Ova) for contact time of ∼1–3 sec and 3 min. *p<0.01, unpaired <i>t</i>-test. Bars indicate mean ± s.e.m (n>10 pairs of cells). For each condition, OT-I T-cells were isolated from >3 independent experiments.</p

B-cells and DCs exhibit a similar phenotype.

<p>B-cells (Red) and DCs (Black) were stained with antibodies against H-2K^b (MHC class I), H-2K^b/Ova (pMHC), CD11c, CD19, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). Filled histograms: isotype controls; Unfilled histograms: staining with antibody. Data are representative of three independent experiments.</p

Contribution of CD80 and CD86 to Interaction Forces and Cytokine Secretion.

<p>(<b>A</b>) Interaction forces; (<b>B</b>) IL-2 and (<b>C</b>) IFN-γ production of DC:T-cell or B:T-cell conjugates in the presence of blocking antibodies against CD80 and/or CD86. DCs or B-cells were pre-pulsed with Ova peptides (+Ova, 10 ng/ml) before antibody blocking. All force measurements were conducted with contact time of 3 min. *p<0.01; unpaired <i>t</i>-test. For each condition, OT-I T-cells were isolated from >3 independent experiments. αCD80: blocking antibody targeting CD80; αCD86: blocking antibody targeting CD86; αCD80/86: αCD80 and αCD86 simultaneously; IgG: isotype controls for both αCD80 and αCD86.</p

IFNγ production by NK cells cultured with BMDCs stimulated with <i>Lm</i> or <i>E. coli</i>.

<p>BMDCs were infected with <i>Lm</i> or <i>E. coli</i> at an MOI of 20 and cultured with syngeneic NK cells for 18 hr. Where indicated, recombinant IFNβ was added to the co-culture one hour after infection. Levels of IFNγ in the culture supernatants were quantified by ELISA. (A) Experimental design. (B) Co-culture experiments. The means ± SDs of three independent experiments are shown. p value <0.01.</p

<i>In vivo</i> IFNβ production during <i>Lm</i> infection.

<p>Mice (n = 5/group) were injected with 1×10⁶ CFU of <i>Lm</i> and <i>E. coli</i>. RNA was extracted at 4 hr, 8 hr and 24 hr p.i. and the IFNβ mRNA was quantified. (A) IFNβ gene expression from total spleen at the time points indicated. (B) IFNβ gene expression in CD11c⁺ and CD11c⁻ cells purified from total spleen. (C) IFNβ gene expression in total spleen from mice infected with <i>E. coli</i> (1×10⁶ CFU) as a positive control. The housekeeping gene <i>PPIA</i> was used as a reference to normalize data. Data shown are representative of at least three independent experiments.</p

Type I IFN production by <i>Lm</i>-infected DCs.

<p>(A) D1 cells were infected with <i>Lm</i> at an MOI of 40. Supernatants were collected at different time points and IFNβ and IFNα levels were quantified by ELISA. (B) D1 cells were infected with <i>Lm</i> at an MOI of 40. RNA was extracted at different time points and used for qRT-PCR analysis. The fold-increases, relative to β-actin, for IFNα4, IFNα9, IFNα5, IFNα2 are shown. (C) D1 cells were infected with <i>Lm</i>, and the RNA was extracted and analyzed by RT-PCR. IFNα6 and IFNα1 mRNA levels are shown. Data shown are representative of at least three independent experiments.</p

NK cell activation in the spleen of mice infected with <i>Lm</i>.

<p>Mice (n = 5/group) were infected with <i>Lm</i> (1×10⁶ CFU)± IFNβ. Spleens were removed five hours after infection and NK cell activation was evaluated. (A) Intracellular staining for IFNγ in DX5-positive cells. (B) Splenocytes were co-cultured with YAC-1 cells and the percentage of target cell lysis was determined after three hours; p-value <0.01. The mean of three independent experiments is shown.</p

Induction of type I IFN genes.

<p>Samples derived from two biological replicates per time point were used for subsequent GeneChip probe arrays. (A) Heat map of the time course of IRG expression in DCs after exposure to <i>Lm</i>. (B) Heat map of the time courses of IFNβ and IFNα gene expression. RNAs were collected at different time points and the gene expression levels were measured by microarray analysis. Heat maps were generated with the GeneSpring hierarchical clustering algorithm. Data show mean fold-changes normalized to the pre-exposure 0 hr time point.</p