title: Probing the applicability of autotransporter based surface display with the EstA autotransporter of Pseudomonas stutzeri A15.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Based 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.
  
  
  CONCLUSIONS
  Since 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.
authors.affiliation: Centre of Microbial and Plant Genetics, KU Leuven, Kasteelpark Arenberg 20, 3001, Heverlee, Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Nicolay
authors.forename: Lynn
authors.initials: L
authors.lastname: Lemoine
authors.forename: Elke
authors.initials: E
authors.lastname: Lievens
authors.forename: Sam
authors.initials: S
authors.lastname: Balzarini
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Stijn
authors.initials: S
authors.lastname: Spaepen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D044223:Pseudomonas stutzeri / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria