1. Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
- Author
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Maaike Schillemans, Peter van Ulsen, Wouter S. P. Jong, Joen Luirink, Corinne M. ten Hagen-Jongman, Graduate School, ACS - Microcirculation, Molecular Microbiology, AIMMS, LaserLaB - Molecular Biophysics, and LaserLaB - Analytical Chemistry and Spectroscopy
- Subjects
0301 basic medicine ,Physiology ,Cell Membranes ,lcsh:Medicine ,Biochemistry ,Membrane Fusion ,law.invention ,Cell membrane ,Cell Fusion ,law ,Medicine and Health Sciences ,Urea ,lcsh:Science ,Multidisciplinary ,biology ,Membrane transport protein ,Chemistry ,Organic Compounds ,Escherichia coli Proteins ,Proteases ,Recombinant Proteins ,Cell biology ,Enzymes ,Body Fluids ,medicine.anatomical_structure ,Blood ,Physical Sciences ,Recombinant DNA ,Cellular Structures and Organelles ,Anatomy ,Bacterial outer membrane ,Research Article ,Cell Physiology ,030106 microbiology ,Protein domain ,03 medical and health sciences ,medicine ,Escherichia coli ,Secretion ,lcsh:R ,Cell Membrane ,Organic Chemistry ,Chemical Compounds ,Membrane Transport Proteins ,Biology and Life Sciences ,Membrane Proteins ,Proteins ,Cell Biology ,Blood Serum ,Outer Membrane Proteins ,Protein tertiary structure ,030104 developmental biology ,biology.protein ,Enzymology ,lcsh:Q ,Physiological Processes ,Immune Serum ,Autotransporters - 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.
- Published
- 2017