Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain

Abstract

Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans, was used to express the light chain variable domain of the human κIO8/O18 germline antibody in the periplasm of E. coli. The expressed protein was purified in good yield (70 mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag. Circular dichroism spectroscopy was used to determine if the secondary and tertiary structures of the protein and its thermal stability corresponded to those of the native folded protein. The expressed and purified protein was indeed properly folded and exhibited a reasonable thermal transition temperature of 53°C. These results indicate that the amicyanin signal sequence may be particularly useful for prokaryotic expression of proteins which are prone to mis-folding, aggregation or formation of inclusion bodies, all of which were circumvented in this study.

Zinc-Dependent Transcriptional Regulation in Paracoccus denitrificans

“…For thermal stability experiments, the intensity at 208 nm was monitored from 25 to 90°C every 0.2°C with a constant heating rate of 1.0°C/min. The fraction of folded protein at a given temperature T was determined using the following equation (Dow et al, 2015): $\begin{array}{c}left\text{Fraction Folded}=\frac{{\theta }_T-{\theta }_{25}}{{\theta }_{25}-{\theta }_{90}}\end{array}$ Values of θ at 25 and 90°C were chosen to correspond to 100 and 0% folded states, respectively. Spectra were fit to estimate secondary structure contributions on the online server Dichroweb (Whitmore and Wallace, 2004) using the CDSSTR algorithm (Sreerama and Woody, 2000).…”

Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

“…For thermal stability experiments, the wavelength at 223 nm was monitored from 25 to 90°C every 0.2°C with a constant heating rate of 1.0°C/min. The fraction of folded protein at a given temperature T was determined using the following equation (38).…”

Recombinant protein production data after expression in the bacterium Escherichia coli

“…Expression of recombinant proteins in the cellular periplasm has certain advantages since it contains less proteases [8] , and provides the proper conditions for the formation of disulfide bonds [9] . If the protein of interest is tagged with the CusFp or SmbPp constructs that include the signal sequences, then the target protein is exported to the periplasm.…”