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

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

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

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

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