Figure 2: Expression of mouse-human hybrid cyclonals.
From: Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria
(a) Antigen-binding activity for mouse, mFab–hFc hybrid, and chimeric anti-MBP cyclonals. ELISA signals (Abs492) for mouse cyclonal and empty plasmid control (light-green triangle) samples in cell lysates were obtained with anti-mouse antibodies; mFab/hFc cyclonal, chimeric cyclonal and corresponding empty plasmid control (dark-green star) were detected in cell lysates with anti-human Fc antibodies. Data is expressed as the mean±s.e.m. of biological triplicates. (b) Soluble anti-MBP cyclonal in the mFab–hFc format was purified from cell lysates prepared from SHuffle by protein-A affinity chromatography and analysed by western blot under non-reducing (left panel) and reducing (right panel) conditions. Arrows indicate fully assembled cyclonal as well as other intermediate species. The percentage of fully assembled heterotetrameric product among all products was 63±5% (left panel) as determined by densitometry analysis. (c) Representative SEC analysis of protein-A-purified anti-MBP cyclonal in the mFab/hFc format performed using a Superdex 200 10/300 GL gel filtration column. Heterotetramer and other intermediate products are labelled.
