Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)
Fig 1
Expression optimization of AChBP and DUB in E. coli.
Protein expression levels in the soluble fractions at (1) 37°C, (2) 25°C and (3) 16°C and insoluble fractions (inclusion body) at (4) 37°C, (5) 25°C and (6) 16°C using the (i) IPTG and (ii) auto induction methods were analyzed for (A) Ls-AChBP, (B) Ac-AChBP and (C) DUB using western blots. Anti-His tag mouse monoclonal antibody at a dilution of 1:3000 in the blocking solution was used as the primary antibody Fluorescently labelled goat anti-mouse IgG (H+L) Alexa Fluor 680 at a dilution of 1:2500 was used as the secondary antibody. The western blots were imaged with an Odyssey Infrared Imaging System (Licor® Biosciences) at a wavelength of 700 nm and 2.0 intensity level. Black arrows indicate expected bands for AChBPs and DUB. (D) Protein expression level in the soluble fraction under the different induction methods and temperatures were quantified for (i) Ls-AChBP and (ii) DUB. Ac-AChBP was not detected in the soluble fraction and therefore not quantified.