A structurally informed autotransporter platform for efficient heterologous protein secretion and display.

W.S.P. Jong, Z. Soprova, K. de Punder, C.M. ten Hagen-Jongman, S. Wagner, J.W. de Gier, P. Andersen, N.N. van der Wel, S. Luirink

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Background: 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 ß-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. Results: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a ß-helical core structure (ß-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 ß-stem appeared unstable after translocation, demonstrating the importance of an intact ß-stem. By interrupting the cleavage site between passenger and ß-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. Conclusions: 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.
LanguageEnglish
JournalMicrobial Cell Factories
Volume11
Issue number85
Early online date18 Jun 2012
DOIs
Publication statusPublished - 2012

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Hemoglobin
Hemoglobins
Peptide Hydrolases
Display devices
Proteins
Recombinant proteins
Vaccines
Recombinant Proteins
Escherichia coli
Bacteria
Crystal structure
Tuberculosis Vaccines
Endopeptidase K
Synthetic Vaccines
Salmonella
Gene encoding
Salmonella typhimurium
Type V Secretion Systems
Gram-Negative Bacteria
Mycobacterium tuberculosis

Cite this

Jong, W.S.P. ; Soprova, Z. ; de Punder, K. ; ten Hagen-Jongman, C.M. ; Wagner, S. ; de Gier, J.W. ; Andersen, P. ; van der Wel, N.N. ; Luirink, S. / A structurally informed autotransporter platform for efficient heterologous protein secretion and display. In: Microbial Cell Factories. 2012 ; Vol. 11, No. 85.
@article{8bdcc79a05cb4f43bcfc1cc6d89e209f,
title = "A structurally informed autotransporter platform for efficient heterologous protein secretion and display.",
abstract = "Background: 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 {\ss}-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. Results: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a {\ss}-helical core structure ({\ss}-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 {\ss}-stem appeared unstable after translocation, demonstrating the importance of an intact {\ss}-stem. By interrupting the cleavage site between passenger and {\ss}-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. Conclusions: 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.",
author = "W.S.P. Jong and Z. Soprova and {de Punder}, K. and {ten Hagen-Jongman}, C.M. and S. Wagner and {de Gier}, J.W. and P. Andersen and {van der Wel}, N.N. and S. Luirink",
year = "2012",
doi = "10.1186/1475-2859-11-85",
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Jong, WSP, Soprova, Z, de Punder, K, ten Hagen-Jongman, CM, Wagner, S, de Gier, JW, Andersen, P, van der Wel, NN & Luirink, S 2012, 'A structurally informed autotransporter platform for efficient heterologous protein secretion and display.', Microbial Cell Factories, vol. 11, no. 85. https://doi.org/10.1186/1475-2859-11-85

A structurally informed autotransporter platform for efficient heterologous protein secretion and display. / Jong, W.S.P.; Soprova, Z.; de Punder, K.; ten Hagen-Jongman, C.M.; Wagner, S.; de Gier, J.W.; Andersen, P.; van der Wel, N.N.; Luirink, S.

In: Microbial Cell Factories, Vol. 11, No. 85, 2012.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - A structurally informed autotransporter platform for efficient heterologous protein secretion and display.

AU - Jong, W.S.P.

AU - Soprova, Z.

AU - de Punder, K.

AU - ten Hagen-Jongman, C.M.

AU - Wagner, S.

AU - de Gier, J.W.

AU - Andersen, P.

AU - van der Wel, N.N.

AU - Luirink, S.

PY - 2012

Y1 - 2012

N2 - Background: 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 ß-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. Results: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a ß-helical core structure (ß-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 ß-stem appeared unstable after translocation, demonstrating the importance of an intact ß-stem. By interrupting the cleavage site between passenger and ß-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. Conclusions: 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.

AB - Background: 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 ß-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. Results: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a ß-helical core structure (ß-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 ß-stem appeared unstable after translocation, demonstrating the importance of an intact ß-stem. By interrupting the cleavage site between passenger and ß-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. Conclusions: 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.

U2 - 10.1186/1475-2859-11-85

DO - 10.1186/1475-2859-11-85

M3 - Article

VL - 11

JO - Microbial Cell Factories

T2 - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

IS - 85

ER -