Difference between revisions of "Part:BBa K633001"

Revision as of 01:09, 19 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 where the passenger enzymes retain their hydrolytic activities after being anchored on the outer surface of E. coli cells. (Becker, Theile, Heppeler & Michalczyk, 2005)

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

Three Dimensional Structure of the "Esta" Membrane Protein, including its esterease translocated domain.

However, we used only its transmembranal domain, without its functional esterease. This approach enables the exchange of different small proteins.
By the use of restriction enzymes, NheI and SacI, according to our results one can insert another enzyme ready to be displayed outside the outer membrane in E. coli.

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]

@@ Line 3: / Line 3: @@
 <br>
 <br>
-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.
+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).
+<br>
+<br>
+The successful cell-surface display of lipases as fusion proteins to an inactive variant of EstA has already been described where the passenger enzymes retain their hydrolytic activities after being anchored on the outer surface of E. coli cells. (Becker, Theile, Heppeler & Michalczyk, 2005)
 <br><br>
-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.
+An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes.
-<br><br>
+ <br>
-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.
-<br><br>
-[[Image:Esta_autotransporter.png]]
 <br>
+<center>[[Image:Esta_autotransporter.png]]</center>
 Three Dimensional Structure of the "Esta" Membrane Protein, including its esterease translocated domain.
 <br>
-'''However, for this part we used only its transmembranal domain, without its functional esterease. This approach enables the exchange of different small proteins.'''
+<br>
+However, we used only its transmembranal domain, without its functional esterease. This approach enables the exchange of different small proteins.
-<br><br>
+<br>
-'''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.
+By the use of restriction enzymes,   NheI and SacI, according to our results one can insert another enzyme ready to be displayed outside the outer membrane in E. coli.
-(http://www.sciencedirect.com/science/article/pii/S0014579305000748)'''
+<br>
+<br>
+'''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
+'''
 <br><br>
 '''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'''