%0 Journal Article
%T A structurally informed autotransporter platform for efficient heterologous protein secretion and display
%A Wouter SP Jong
%A Zora Soprova
%A Karin de Punder
%A Corinne M ten Hagen-Jongman
%A Samuel Wagner
%A David Wickstr£¿m
%A Jan-Willem de Gier
%A Peter Andersen
%A Nicole N van der Wel
%A Joen Luirink
%J Microbial Cell Factories
%D 2012
%I BioMed Central
%R 10.1186/1475-2859-11-85
%X Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a ¦Â-helical core structure (¦Â-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 ¦Â-stem appeared unstable after translocation, demonstrating the importance of an intact ¦Â-stem. By interrupting the cleavage site between passenger and ¦Â-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.
%K Autotransporter
%K Type V secretion
%K Hemoglobin protease
%K Extracellular expression
%K Surface display
%K Live vaccine
%U http://www.microbialcellfactories.com/content/11/1/85/abstract