Optimization of Haemophilus influenzae adhesin transmembrane domain expression in Escherichia coli

Abstract

To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability. As a result, the amount of purified HiaTD reached a plateau at 2 h postinduction. Although expression without the signal sequence provides a large amount of proteins as inclusion bodies in some membrane proteins, HiaTD expressed without a signal sequence was not observed as inclusion bodies and seemed to be assembled into the outer membrane during or after cell lysis.

Keywords: Outer membrane protein; Recombinant protein expression; Trimeric autotransporter.