Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface

Abstract

Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

“…A panel of cell surface scaffold modules were chosen based on their ability to direct passenger proteins to the E. coli outer membrane. These included cytolysin ClyA (18), hybrid protein Lpp-OmpA (35), and the autotransporter β -domains derived from the N-terminus of intimin (Int) (36) and the C-termini of adhesin involved in diffuse adherence (AIDA-I), antigen-43 (Ag43), hemoglobin-binding protease (Hbp), and immunoglobulin A protease (IgAP) (37).…”

Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras

“…In earlier studies, we have reported on the limited tolerance of Hbp to transport folded heterologous protein domains across the outer membrane where such DTS constructs showed a decreased secretion efficiency when compared to wild-type Hbp or efficiently-secreted derivatives [ 13 , 16 , 41 ]. Examples of DTS inserts are the calmodulin domain that forms a stable fold in the presence of calcium ions [ 16 ] and a single-chain antibody domain that includes two disulfide bonds [ 14 ]. To systematically investigate the limits of Hbp secretion, we previously constructed a DTS Hbp with an α-helical hairpin formed by two stable α-helices derived from ribosomal protein L9 of Bacillus stearothermophilus constricted into an hairpin through a disulfide bond formed between cysteine residues engineered at positions 707 and 712 of the Hbp passenger (Fig.…”

“…These small domains (15 kDa) are known to fold in the periplasm and include one disulphide bond. Fusing GFPnb to HbpD has been shown to impair expression and surface display [ 14 , 52 ]. We tested the effect of co-expression of BAM on expression and display of DTS HbpD-GFPnb cloned into pLEMO.…”

“…Previously, we have attempted to expand the secretion capacity of the Hbp system by testing adaptations to the β-barrel domain and by replacing it for the β-barrel domain of another autotransporter [ 14 , 39 ] but with limited success. Here we examined whether increased expression of generic components of the secretion route of autotransporters can improve secretion of overproduced and difficult-to-secrete (DTS) recombinant Hbp chimeras.…”

Neisseria gonorrhoeae NGO2105 Is an Autotransporter Protein Involved in Adhesion to Human Cervical Epithelial Cells and in vivo Colonization

“…Various specific β-barrels of autotransporters have been shown to secrete recombinant proteins to the cell surface, but the transport efficiencies of different β-barrels are different. The Hbp passenger domain is often used as a transport target to evaluate the transport capacity of the β-barrels of autotransporters (Jong et al, 2018). To investigate whether the NGO2105 β-barrel can secrete the heterologous passenger domain, we fused a small Myc tag and Hbp passenger domain upstream of the β-barrel of NGO2105.…”