A structurally informed autotransporter platform for efficient heterologous protein secretion and display
1 Section Molecular Microbiology, Department of Molecular Cell Biology, Faculty of Earth and Life Sciences, VU University, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands
2 Xbrane Bioscience, SE-111 45, Stockholm, Sweden
3 The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, 1066 CX, Amsterdam, The Netherlands
4 Inter-Faculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), 72076, Tübingen, Germany
5 Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden
6 Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
Microbial Cell Factories 2012, 11:85 doi:10.1186/1475-2859-11-85Published: 18 June 2012
The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.
Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.
We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.