Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

The diagnostic targeting of a carbohydrate virulence factor from M.Tuberculosis

10.1038/srep10281Scientific Reports

Large scale purification of bacterial IgG.

<p>(A) Non-reducing coomassie gel of peak fractions from Protein A HPLC purification of cell lysate. Left panel: 4G2 fractions 5 to 11 (F5 to F11). Right panel ET149 fractions 10 to 16 (F10 to F16). 30 µl of each fraction was run on the gel. A sample of the wash (LW) was run and shows no contaminants present indicating the column was sufficiently washed to remove non-binding proteins. Fully assembled IgGs (2H2L) are indicated. (B). Reducing coomassie gels (4G2-F7, ET149-F12 & 13) and adjacent immunoblots (I) showing representative fractions from each Protein A elution (indicated with * on panel A). 30 µl of each fraction was loaded for coomassie and 3.75 µl for immunoblot. Blots were probed with both anti-IgG Fc and anti-Kappa chain polyclonals showing that majority of protein bands in the fraction are neither IgG heavy chain nor light chain. A separate reducing coomassie gel shows 2 µg of bacterial IgG pooled eluate (EL) after Protein L purification showing successful removal of the contaminating proteins and degraded heavy chain fragments. The equivalent amount of mammalian cell culture-derived IgG (IgG) was loaded for comparison. Individual heavy (HC) and light (LC) chains are indicated although the light chain for ET149 appears as two separate species.</p

Comparison between bacterial and mammalian IgG.

<p>(A). Non-reducing SDS-PAGE of 2 µg of pooled bacterial IgG eluate after protein L purification (EL) and mammalian cell culture-derived IgG (IgG) indicates that the majority of purified IgG was fully assembled IgG (2H2L) although partially assembled IgG (2H1L, 1H1L) are also present. (B). Direct binding ELISA of serially diluted bacterial (-▪-)- and mammalian cell culture (-•-)-derived IgG against Dengue serotype-2 virus and epsilon toxin showing similar binding curves. Binding of antibody was detected using anti-Human IgG Fc-HRPO as the secondary antibody.</p

Small scale optimization of bacterial IgG expression.

<p>(A-E) Left and middle panels: Non-reducing immunoblot of 7.5 µl clarified cell lysate from small scale overnight expression of bacterial IgG in shaking culture. All blots were probed with anti-Human Fc-HRPO and adjusted to ensure equal intensity. Arrows indicate fully-assembled IgG. Right panel: Direct binding ELISA indicating levels of functional IgG. Background binding signal was negligible for all cell lysate samples at the indicated dilution or neat. Error bars were calculated from average of three or four observations. Expressed antibodies were 4G2 (A), PA38 (B), PA64 (C), ET21 (D), ET149 (E). (F) Variation in wet cell mass for all five antibodies under different induction conditions. Mass is indicated in mg</p

Construction of tet promoter bacterial IgG expression plasmid.

<p>pASK-IBA2 plasmid using a <i>tet</i> promoter was used as the backbone expression vector. The appropriate restriction sites for cloning in of the light and variable heavy chains, leader sequences (OmpA, PelB) and the constant heavy chain sequence (CH) were added. Light (LC) and variable chains (VH) were cloned in as a complete construct together with the intercistronic ribosomal binding site (RBS) from the phage display vector or as separate constructs from the 4G2 mammalian IgG expression vector.</p

Initial bacterial IgG expression and periplasmic extraction.

<p>(A and B) Non-reducing and reducing immunoblot of periplasmic extract from overnight expression of 4G2 in HB2151 (HB) and BL21(DE3) (BL) <i>E. coli</i> strains, respectively. All blots were probed with anti-Human Fc-HRPO.</p

Virus-specific T lymphocytes home to the skin during natural dengue infection

10.1126/scitranslmed.aaa0526Science Translational Medicine727

Enhanced neutralizing antibody titers and Th1 polarization from a novel Escherichia coli derived pandemic influenza vaccine.

International audienceInfluenza pandemics can spread quickly and cost millions of lives; the 2009 H1N1 pandemic highlighted the shortfall in the current vaccine strategy and the need for an improved global response in terms of shortening the time required to manufacture the vaccine and increasing production capacity. Here we describe the pre-clinical assessment of a novel 2009 H1N1 pandemic influenza vaccine based on the E. coli-produced HA globular head domain covalently linked to virus-like particles derived from the bacteriophage Qβ. When formulated with alum adjuvant and used to immunize mice, dose finding studies found that a 10 µg dose of this vaccine (3.7 µg globular HA content) induced antibody titers comparable to a 1.5 µg dose (0.7 µg globular HA content) of the licensed 2009 H1N1 pandemic vaccine Panvax, and significantly reduced viral titers in the lung following challenge with 2009 H1N1 pandemic influenza A/California/07/2009 virus. While Panvax failed to induce marked T cell responses, the novel vaccine stimulated substantial antigen-specific interferon-γ production in splenocytes from immunized mice, alongside enhanced IgG2a antibody production. In ferrets the vaccine elicited neutralizing antibodies, and following challenge with influenza A/California/07/2009 virus reduced morbidity and lowered viral titers in nasal lavages

Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine

Abstract Cell mediated immunity plays a vital role in defense against influenza infection in humans. Less is known about the role of vaccine-induced cell mediated immunity and the cytokine responses elicited. We measured CD4+ and CD8+ T-cell reactivity in human subjects following vaccination with licensed trivalent influenza vaccine and a novel virus-like particle based vaccine. We detected influenza-specific CD4+ T-cell responses following vaccination with the licensed trivalent influenza vaccine and found that these correlated with antibody measurements. Administration of the novel virus-like particle based vaccine elicited influenza-specific CD4+ and CD8+ T-cell responses and the induction of the cytokines IFN-γ, IL-17A, IL17F, IL-5, IL-13, IL-9, IL-10 and IL-21. Pre-existing cytokine responses influenced the profile of the cytokine response elicited by vaccination. In a subset of individuals the VLP vaccine changed pre-vaccination production of type 2 cytokines such as IL-5 and IL-13 to a post-vaccination type 1 cytokine signature characterized by IFN-γ. A transcriptional signature to vaccination was found to correlate with antibody titer, IFN-γ production by T-cells and expression of a putative RNA helicase, DDX17, on the surface of immune cells