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

<div><p>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 V_HH 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 V_HH 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.</p></div

Expression and surface-accessibility of Hbp-β domain fusions.

<p><b>(A)</b> Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing wild-type Hbp and Hbp(<i>Spe</i>I). <b>(B)</b> Coomassie-stained gel of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 <i>degP</i>::S210A (right panel) expressing Hbp-β-domain fusions. <b>(C)</b> Coomassie-stained gel of whole cell lysates of cells of MC1061 expressing the Hbp-β-domain fusions incubated with proteinase K, either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-).The positions of bands representing unprocessed Hbp passenger-β-domain fusions (*), and processed Hbp passenger (●) and Hbpβ (▲) are indicated. The latter are only detected for Hbp(<i>Spe</i>I). In panel C, the prominent ~79-kDa of Hbp-Δβcleavage degradation product is indicated by (#), the position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis.</p

Expression and surface-accessibility of V_HH-β domain fusions.

<p><b>(A)</b> Cartoon of the structure of the V_HH domain fused to the eight β-domain constructs. The disulfide bond that stabilizes the secondary structure and is likely formed in the periplasm is highlighted in blue. <b>(B)</b> Coomassie-stained SDS-PAGE of whole cell lysates (c) and culture supernatants (m) of MC1061 (left panel) or MC1061 <i>degP</i>::S210A (right panel) cells expressing V_HH-β-domain fusions. Indicated are the detectable V_HH-β-domain fusions (*). <b>(C)</b> Western blots incubated with α-Myc of whole cell lysates (c) and culture supernatants (m) of MC1061 cells expressing the V_HH-β-domain fusions (*). The expected position of a processed V_HH-Myc fusion of 18 kDa is indicated on the left. Note that the right panel is derived from a different blot. Compare the results also with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191622#pone.0191622.g002" target="_blank">Fig 2S</a> that shows that blots of constructs expressed in <i>E</i>. <i>coli</i> strains DHB4 and DHBA yielded a very similar detection pattern. <b>(D)</b> Coomassie-stained SDS-PAGE gels of whole cell lysates of cells of MC1061 cells expressing the V_HH-Hbpβ or V_HH-IgAPβ(1245) (*) incubated with 100 μg/ml proteinase K (pk) or with buffer (-) for 30 min at 37 °C. The position of the proteinase K bands is indicated by the closed arrowhead, whereas the open arrowheads indicate the control bands used for densitometric analysis (<b>E)</b> Microscopic images of MC1061 cells expressing V_HH-Hbpβ (right panels) or V_HH-IgAPβ(1245) (left panels). The top panels are phase-contrast images, the lower panels are immunofluorescent images. Contours of cells are visible due to background autofluoresence.</p

Expression, outer-membrane insertion and folding of Myc-β domain fusions.

<p><b>(A)</b> Coomassie-stained SDS-PAGE gel (left panel) and Western blot incubated with antiserum against the myc tag (α-Myc; right panel) to detect expression of Myc-β-domain fusions in MC1061 cells. Indicated are Myc-β-domain fusions (*), degradation products (●) and bands of higher apparent molecular wheight (▲). <b>(B)</b> Western blots incubated with antisera against the myc tag (α-Myc), the Hbpβ (α-Hbpβ) and IgAPβ (α-IgAPβ) to assess insertion into the outer membrane. CE, isolated cell envelopes; P, pellet fractions of cell envelopes after Urea extraction; S, supernatant fractions of cell envelopes after Urea extraction. <b>(C)</b> Western blots incubated with α-Myc to assess heat-modifiability of membrane-inserted Myc-β-domain fusions. Samples of isolated cell envelopes were either heated at 98 °C (h) or kept at room temperature (rt). The positions of bands representing denatured proteins (*) running at the position expected from the calculated molecular wheight and folded proteins (#) running at a lower position are indicated.</p