Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15

Toon Nicolay,Stijn Spaepen,Lynn Lemoine,Elke Lievens,Sam Balzarini,Jos Vanderleyden

doi:10.1186/1475-2859-11-158

Abstract

BackgroundAutotransporters represent a widespread family of secreted proteins in Gram-negative bacteria. Their seemingly easy secretion mechanism and modular structure make them interesting candidates for cell surface display of heterologous proteins. The most widely applied host organism for this purpose is Escherichia coli. Pseudomonas stutzeri A15 is an interesting candidate host for environmentally relevant biotechnological applications. With the recently characterized P. stutzeri A15 EstA autotransporter at hand, all tools for developing a surface display system for environmental use are available. More general, this system could serve as a case-study to test the broad applicability of autotransporter based surface display.ResultsBased on the P. stutzeri A15 EstA autotransporter β-domain, a surface display expression module was constructed for use in P. stutzeri A15. Proof of concept of this module was presented by successful surface display of the original EstA passenger domain, which retained its full esterase activity. Almost all of the tested heterologous passenger domains however were not exposed at the cell surface of P. stutzeri A15, as assessed by whole cell proteinase K treatment. Only for a beta-lactamase protein, cell surface display in P. stutzeri A15 was comparable to presentation of the original EstA passenger domain. Development of expression modules based on the full-length EstA autotransporter did not resolve these problems.ConclusionsSince only one of the tested heterologous passenger proteins could be displayed at the cell surface of P. stutzeri A15 to a notable extent, our results indicate that the EstA autotransporter cannot be regarded as a broad spectrum cell surface display system in P. stutzeri A15.

Highlights

Autotransporters represent a widespread family of secreted proteins in Gram-negative bacteria
The designed expression modules, integrated in the pHERD26T backbone, contain a transport unit consisting of the β-barrel and the α-helical linker of P. stutzeri A15 EstA as derived from the structural model
The 50 untranslated region (UTR) of pEstAβL is identical to the 50 UTR of pHERD26T-estA, which expresses the EstA AT, and was expected to have similar expression levels as the latter

Summary

Introduction

Autotransporters represent a widespread family of secreted proteins in Gram-negative bacteria Their seemingly easy secretion mechanism and modular structure make them interesting candidates for cell surface display of heterologous proteins. With the recently characterized P. stutzeri A15 EstA autotransporter at hand, all tools for developing a surface display system for environmental use are available. Apart from this, ATs are mainly of interest because replacement of the passenger domain with a heterologous protein makes them suitable candidates for cell surface display, a process termed autodisplay. Amongst others, it has been applied in vaccine development, library screening and whole-cell biocatalysis [6]. Often this system is regarded as a universal and broadly applicable cell surface display system posing minimal requirements to the passenger domain [6,8]

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