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

Wouter S.P. Jong*, Maaike Schillemans, Corinne M. ten Hagen-Jongman, Joen Luirink, Peter van Ulsen

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Article numbere0191622
JournalPLoS ONE
Volume13
Issue number2
DOIs
Publication statusPublished - 7 Feb 2018

Fingerprint Dive into the research topics of 'Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface'. Together they form a unique fingerprint.

Cite this