Difference between revisions of "Part:BBa K633001"
 
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EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes.
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EstA is an outer membrane-anchored esterase from ''Pseudomonas aeruginosa''. It has autotransporter activities that reside in its catalytic N-terminal domain, the C-terminal domain formes a β-barrel-like structure. EstA is inserted in the bacterial outer membrane where the N-terminal domain is translocated outside the outer membrane. (Becker, Theile, Heppeler & Michalczyk, 2005).
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The successful cell-surface display of lipases as fusion proteins to an inactive variant of EstA has already been described. Passenger enzymes in that fusion retain their hydrolytic activities after being anchored on the outer surface of ''Escherichia coli'' cells. (Becker, Theile, Heppeler & Michalczyk, 2005)
 
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EstA is an autotransporter protein which consists of an N-terminal domain harboring the catalytic activity and a C-terminal domain forming a b-barrel-like structure inserted into the bacterial outer membrane which mediates the translocation of the N-terminal domain.
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An inactive EstA variant was used as an anchoring motif for the ''E. coli'' cell-surface display of lipolytic enzymes.
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<center>[[Image:Esta_autotransporter_real.png]]</center>
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Fig. 1. Overall structure of the full-length EstA AT. (a) Backbone representation viewed from the side, with the protein
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colored by a gradient from blue at the N-terminus to red at the C-terminus. Strands S1 and S12 are indicated, with an
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arrow highlighting the connection between the central α-helix and strand S1. (b) Backbone view 90° rotated within the
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plane of the membrane relative to (a), with helices colored red, β-strands colored green, and loops colored gray. The
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catalytic triad residues are shown as blue stick models. The approximate location of the OM core is indicated by
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horizontal lines. (c) Surface view of EstA from the side, with the β-barrel domain colored green and the passenger domain
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colored red. (d) Stereo view of the EstA passenger from the extracellular side, colored as a rainbow from dark blue at the
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N-terminus to dark red at the C-terminus. The numbers are those for the central residue of the α-helix. For clarity, the
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loops have been smoothed. All figures were made using PyMOL.18
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Three Dimensional Structure of the "EstA" Membrane Protein, including its esterease translocated domain.
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We used only its transmembranal domain, without its functional esterease. This approach enables the exchange of different small proteins.
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References:
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Becker, S., Theile, S., Heppeler, N., & Michalczyk, A. (2005). A generic system for the escherichia coli cell-surface display of lipolytic enzymes. FEBS Letters, 579(5), 1177-1182. Retrieved from http://www.sciencedirect.com/science/article/pii/S0014579305000748
 
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The successful cell-surface display of lipases as fusion proteins to an inactive variant of EstA has already been described in several papers, where the passenger enzymes retain their hydrolytic activities after being anchored on the outer surface of E. coli cells.
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Van Den Berg, B. (2010). Crystal structure of a full-length autotransporter. Journal of Molecular Biology, 396(3), 627-633. Elsevier Ltd. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20060837
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[[Image:Esta_autotransporter.png]]
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''Stefan Becker, Sebastian Theile, Nele Heppeler, Anja Michalczyk, Alexander Wentzel, Susanne Wilhelm, Karl-Erich Jaeger, Harald Kolmar, A generic system for the Escherichia coli cell-surface display of lipolytic enzymes, FEBS Letters, Volume 579, Issue 5, 14 February 2005, Pages 1177-1182, ISSN 0014-5793, 10.1016/j.febslet.2004.12.087.
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(http://www.sciencedirect.com/science/article/pii/S0014579305000748)''
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 00:38, 20 October 2011

Linker + EstA Membrane Protein

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. It has autotransporter activities that reside in its catalytic N-terminal domain, the C-terminal domain formes a β-barrel-like structure. EstA is inserted in the bacterial outer membrane where the N-terminal domain is translocated outside the outer membrane. (Becker, Theile, Heppeler & Michalczyk, 2005).

The successful cell-surface display of lipases as fusion proteins to an inactive variant of EstA has already been described. Passenger enzymes in that fusion retain their hydrolytic activities after being anchored on the outer surface of Escherichia coli cells. (Becker, Theile, Heppeler & Michalczyk, 2005)

An inactive EstA variant was used as an anchoring motif for the E. coli cell-surface display of lipolytic enzymes.

Esta autotransporter real.png


Fig. 1. Overall structure of the full-length EstA AT. (a) Backbone representation viewed from the side, with the protein colored by a gradient from blue at the N-terminus to red at the C-terminus. Strands S1 and S12 are indicated, with an arrow highlighting the connection between the central α-helix and strand S1. (b) Backbone view 90° rotated within the plane of the membrane relative to (a), with helices colored red, β-strands colored green, and loops colored gray. The catalytic triad residues are shown as blue stick models. The approximate location of the OM core is indicated by horizontal lines. (c) Surface view of EstA from the side, with the β-barrel domain colored green and the passenger domain colored red. (d) Stereo view of the EstA passenger from the extracellular side, colored as a rainbow from dark blue at the N-terminus to dark red at the C-terminus. The numbers are those for the central residue of the α-helix. For clarity, the loops have been smoothed. All figures were made using PyMOL.18 Three Dimensional Structure of the "EstA" Membrane Protein, including its esterease translocated domain.

We used only its transmembranal domain, without its functional esterease. This approach enables the exchange of different small proteins.



References:


Becker, S., Theile, S., Heppeler, N., & Michalczyk, A. (2005). A generic system for the escherichia coli cell-surface display of lipolytic enzymes. FEBS Letters, 579(5), 1177-1182. Retrieved from http://www.sciencedirect.com/science/article/pii/S0014579305000748

Van Den Berg, B. (2010). Crystal structure of a full-length autotransporter. Journal of Molecular Biology, 396(3), 627-633. Elsevier Ltd. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20060837


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 217
  • 1000
    COMPATIBLE WITH RFC[1000]