Team:Berkeley Wetlab/Cell Surface Display Parts

From 2009.igem.org

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[[Image:schematic.jpg|center|500px]]<br>
[[Image:schematic.jpg|center|500px]]<br>
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==Passengers==
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==<center>Passengers</center>==
<font color="#41383C" size="2">Follow any of the links below to see assay information for each of the passengers we made.</font>
<font color="#41383C" size="2">Follow any of the links below to see assay information for each of the passengers we made.</font>
{| cellpadding="25" cellspacing="0"
{| cellpadding="25" cellspacing="0"
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| <html>
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| {{linkedImage|Mgfp5button.png|Team:Berkeley_Wetlab/Passenger:_MGFP}}
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<div align="left">
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[[Team:Berkeley_Wetlab/Passenger: MGFP | '''MGFP-5''']]<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Streptavidin">
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'''The Sticky Protein'''<br>
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  <img width="175" src="https://static.igem.org/mediawiki/2009/2/22/Streptagbutton.png">
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Displaying this sticky peptide would allow you to make an underwater bio-glue!
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</a>
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|| {{linkedImage|Cellulasebutton.png|Team:Berkeley_Wetlab/Passenger:_Cellulases}}
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</div>
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[[Team:Berkeley_Wetlab/Passenger: Cellulases | '''Cellulase''']]<br>
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</html><br>
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'''The Enzyme'''<br>
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[[Team:Berkeley_Wetlab/Passenger: Streptavidin | '''The Tag''']]<br>
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Displaying enzymes that degrade cellulose would allow consolidated bioprocessing for biofuel production!<br>
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A tag that binds the protein streptavidin!<br>
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|| {{linkedImage|Scfvbutton.png|Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv}}
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<br>
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[[Team:Berkeley_Wetlab/Passenger: TypeIII Needle scFv | '''Type III Needle scFv''']]<br>
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|| <html>
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'''The Antibody Fragment'''<br>
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<div align="left">
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Displaying antibody fragments would allow easy pathogen detection!<br>
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Leucine_Zippers">
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  <img width="175" src="https://static.igem.org/mediawiki/2009/d/df/Leucinezipeprsbutton.png">
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</a>
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</div>
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</html><br>
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[[Team:Berkeley_Wetlab/Passenger: Leucine Zippers | '''Binding Peptide #1''']]<br>
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A structural motif that can allow different cell types to recognize and to bind each other!<br>
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|| <html>
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<div align="left">
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<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide">
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  <img width="175" src="https://static.igem.org/mediawiki/2009/a/aa/Ag4.png">
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</a>
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</div>
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</html><br>
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[[Team:Berkeley_Wetlab/Passenger: Ag4 Peptide | '''Binding Peptide #2''']]<br>
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A peptide that reduces silver ions to form a silver precipitate!<br>
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|-
|-
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| <html>
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| {{linkedImage|Ag4.png|Team:Berkeley_Wetlab/Passenger:_Streptavidin}}
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<div align="left">
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[[Team:Berkeley_Wetlab/Passenger: Streptavidin | '''Streptavidin Binding Tag''']]<br>
-
<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_MGFP">
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'''The Tag'''<br>
-
  <img width="175" src="https://static.igem.org/mediawiki/2009/6/63/Mgfp5button.png">
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A tag on the surface of E.coli would allow you to purify bacteria preferentially!<br>
-
</a>
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|| {{linkedImage|Leucinezipeprsbutton.png|Team:Berkeley_Wetlab/Passenger:_Leucine_Zippers}}
-
</div>
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[[Team:Berkeley_Wetlab/Passenger: Leucine Zippers | '''Leucine Zippers''']]<br>
-
</html><br>
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'''The Heterodimeric Binding Peptide'''<br>
-
[[Team:Berkeley_Wetlab/Passenger: MGFP | '''The Sticky Protien''']]<br>
+
Displaying heterodimeric binding peptides would allow you to make heterogeneous bacteria systems!<br>
-
A protein used by mussels to stick to rocks.  An underwater bio-glue!<br>
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|| {{linkedImage|Streptagbutton.png|Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide}}
-
|| <html>
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[[Team:Berkeley_Wetlab/Passenger: Ag4 Peptide | '''Ag4 ''']]<br>
-
<div align="left">
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'''The Silver Binding Peptide'''<br>
-
<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Cellulases">
+
The metal head of our set! Displaying a heavy metal binding peptide would promote bioremediation of heavy metals and aid in synthesis of biomaterials. <br>
-
  <img width="175" src="https://static.igem.org/mediawiki/2009/d/df/Cellulasebutton.png">
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-
</a>
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-
</div>
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-
</html><br>
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-
[[Team:Berkeley_Wetlab/Passenger: Cellulases | '''The Enzyme''']]<br>
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Enzymes that degrade cellulose!<br>
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-
|| <html>
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-
<div align="left">
+
-
<a href="https://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv">
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-
  <img width="175" src="https://static.igem.org/mediawiki/2009/d/d8/Scfvbutton.png">
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-
</a>
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</div>
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-
</html><br>
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[[Team:Berkeley_Wetlab/Passenger: TypeIII Needle scFv | '''The scFv''']]<br>
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An antibody fragment that binds a motif common to enteropathogenic bacteria!<br>
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|}
|}
-
==Spacers==
+
==<center>Spacers</center>==
-
Multiple spacer elements are typically present in natural display systems suggesting that they are important components of cell surface display systems. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized.  We hoped to characterize the effects of the inclusion of spacer elements within our passenger-display design. There were five spacer domains introduced to our surface display system: INP-repeats, beta roll, bet helix, Gly-Ser repeats and GFP-LVA. These elements are further discussed below. <br>
+
Spacer elements are present in most natural display systems, which suggests that they are important components of cell surface display. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized and most people engineer cell surface display devices without structural spacers.  We decided to build a few cell surface display systems with spacer elements in order to begin characterizing their role within engineered cell surface display devices. We introduced five different spacer domains to our surface display system: INP-repeats, beta roll, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below. <br>
-
INP - Repeats-repeated portion of ice nucleation protein (INP) sequence. <br>
+
{|style="background-color:#D2B9D3;" cellpadding="10" cellspacing="0" border="1"
 +
| '''INP-Repeats'''
 +
||Synthetic spacer region composed of repeated portions of ice nucleation protein (INP) sequence.
 +
|-
 +
|'''Beta Roll'''
 +
||Protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.
 +
|-
 +
|'''Beta Helix'''
 +
||Protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.
 +
|-
 +
| '''Gly-Ser Repeats'''
 +
|| Synthetic spacer made from fourteen repeated glycine and serine residues. This spacer is essentially devoid of secondary structure, it is simply a long flexible linker.  We made in order to differentiate between the importance of a natural spacer with secondary/tertiary structure and a long linker between the displayer and its passenger.
 +
|-
 +
|'''GFP-LVA'''
 +
||Modified green fluorescent protein
 +
|}
-
Beta Roll - protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.
+
==<center>Displayers</center>==
-
 
+
A displayer is defined as an outmembrane protein that carries another protein to the extracellular space of the cell.
-
Beta Helix - protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.
+
-
 
+
-
Gly-Ser Repeats - a flexible 14-mer amino acid sequence of glycine and serine residues repeated.
+
-
 
+
-
GFP-LVA -
+
-
 
+
-
==Displayers==
+
-
An outmembrane protein that carries another protein through the outermembrane
+
For successful cell surface display of proteins, there must be an effective protein localization mechanism. Gram-negative bacteria such as E. Coli have two membranes, which present a problem for transporting proteins synthesized in the cytoplasm to the outside of the cell. Various transport schemes exist in gram-negative bacteria to effectively localize proteins to the outermembrane. The most common schemes are TypeI, TypeIII, and TypeV secretion.
For successful cell surface display of proteins, there must be an effective protein localization mechanism. Gram-negative bacteria such as E. Coli have two membranes, which present a problem for transporting proteins synthesized in the cytoplasm to the outside of the cell. Various transport schemes exist in gram-negative bacteria to effectively localize proteins to the outermembrane. The most common schemes are TypeI, TypeIII, and TypeV secretion.
   
   
-
In our display systems, we chose a class of outermembrane proteins called autotransporters that localizes proteins via the TypeV secretion mechanism. Over 700 autotransporters have been sequenced, many of which are used to export virulence factors to the outside of the cell. We decided to harvest this localization system for cell surface display because the outermembrane protein (aka displayer) spontaneously inserts into the outermembrane and pulls the protein it is covalently linked to (aka passenger)into the extracellular space. Moreover, autotransporters are capable of pulling through large proteins, such as enzymes and single-chain variable fragments.
+
In our display systems, we chose a class of outermembrane proteins called autotransporters that localizes proteins via the TypeV secretion mechanism. Over 700 autotransporters have been sequenced, many of which are used to export virulence factors to the outside of the cell. We decided to harvest this localization system for cell surface display because the outermembrane protein (aka displayer) spontaneously inserts into the outermembrane and pulls the protein it is covalently linked to (aka passenger) into the extracellular space. Moreover, autotransporters are capable of pulling through large proteins, such as enzymes and single-chain variable fragments. We have removed the native passengers in our autotransporter constructs and fused heterologous passengers of interest to the N-terminus of these autotransporters.  
[[Image:Autotransporter secretion.png|600px|center]]
[[Image:Autotransporter secretion.png|600px|center]]
-
As depicted in the diagram above, the autotransporter localization begins with localization to the periplasm via the Sec secretion pathway. The translocated protein remains unfolded in the periplasm until it inserts into the outermembrane by forming a beta barrel with its C-terminal 250-300 amino acyl residues. The N-terminus of the protein (containing our passenger of interest) is then pulled through the barrel to the outside of the cell. Passengers of displayers are often cleaved for extracellular secretion. In our systems, however, we cleaved the prepro sequence that signals for peptide cleavage so that our passengers would remain attached to the transmembrane displayer protein.  
+
As depicted in the diagram above, autotransporter transport begins with localization to the periplasm via the Sec secretion pathway. The translocated protein remains unfolded in the periplasm until it inserts into the outermembrane by forming a beta barrel with its C-terminal 250-300 amino acyl residues. The N-terminus of the protein (containing our passenger of interest) is then pulled through the barrel to the outside of the cell. Passengers of displayers are often cleaved for extracellular secretion. In our systems, however, we removed the signal sequence that signals for peptide cleavage so our passengers remain attached to the transmembrane displayer protein.  
-
In constructing our parts, we looked into various autotransporters with different attributes conducive to cell surface display.  
+
In constructing our parts, we looked into a broad range of autotransporters, some well characterized and others putative, to explore the spectrum of display machinery and to establish the functionality of novel autotransporters for cell surface display.  
-
azo1653 AtD (putative) - organism Azoarcus sp. (strain BH72)<br>
+
{|style="background-color:#F9966B;" border="1;"
-
This protein contains one autotransporter domain of the AT-1 family.
+
|'''Azo1653 AtD (putative)'''
-
 
+
||Organism: Azoarcus sp. (strain BH72)<br>
-
OprF AtD - organism Pseudomonas fluorescens<br>
+
Autotransporter type: AT-1 family
-
This outermembrane protein is thought to form an 8-stranded beta barrel in the outermembrane.
+
|-
-
 
+
|'''OprF AtD'''
-
Cl02365 AtD (putative) - organism Neisseria meningitidis<br>
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||Organism: Pseudomonas fluorescens<br>
-
This protein contains one autotransporter domain of the AT-1 family.
+
Structure: an 8-stranded beta barrel in the outermembrane
-
 
+
|-
-
VtaA11 AtD - organism Haemophilus parasuis<br>
+
|'''Cl02365 AtD (putative)'''
-
This protein contains an autotransporter domain of the AT-2 family.
+
||Organism: Neisseria meningitidis<br>
-
 
+
Autotransporter type: AT-1 family
-
Hag AtD - organism Moraxella catarrhalis<br>
+
|-
-
The protein is a 200kDa outermembrane oligomeric protein with beta barrel structure composing of 10 transmembrane beta helices. <br>
+
|'''VtaA11 AtD'''
-
[[Image:Hag autotransporter.jpg|100px|predicted 2D structure]]
+
||Organism: Haemophilus parasuis<br>
-
 
+
Autotransporter type: AT-2 family
-
Pcryo_1225AtD - organism Psychrobacter cryohalolentis
+
|-
-
 
+
|'''Hag AtD'''
-
Hia AtD - organism Haemophilus influenzae <br>
+
||Organism: Moraxella catarrhalis<br>
-
[[Image:Hia ATD.jpg|100px]]
+
Autotransporter type: dimeric family<br>
-
 
+
Structure: 200kDa protein with 10-stranded beta barrel<br>
-
upaG_short - organism Escherichia Coli
+
[[Image:Hag autotransporter.jpg|predicted 2D structure of Hag AtD|150px]]
-
 
+
|-
-
espP(beta) - organism Escherichia coli<br>
+
|'''Pcryo_1225 AtD (putative)'''
-
[[Image:EspP ATD.jpg|100px]]
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||Organism: Psychrobacter cryohalolentis<br>
-
 
+
|-
-
ehaB - organism Escherichia coli
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|''' Hia AtD'''
-
 
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||Organism: Haemophilus influenzae <br>
-
TshA - organism Escherichia coli<br>
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Autotransporter domain: trimeric family<br>
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subgroup1 of autotransporters
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Structure: modular segments containing repeats of structurally distinct domains<br>
-
 
+
[[Image:Hia ATD.jpg|150px]]
-
VirG(IcsA) - organism Shigella flexneri
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|-
-
 
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|'''upaG_short'''
-
YuaQ AtD - organism Escherichia coli
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||Organism: Escherichia Coli<br>
-
 
+
Autotransporter type: trimeric family
-
AIDA-I - organism Escherichia Coli
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|-
-
 
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|'''espP(beta)'''
-
Ag43_short - organism Escherichia Coli MG1655
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||Organism: Escherichia coli<br>
-
 
+
Structure: 12-stranded beta barrel<br>
-
eCPX - circularly permuted OmpX(organism Escherichia Coli)
+
[[Image:EspP ATD.jpg|170px]]
-
This protein is an enhanced CPX variant that is located in the outermembrane that joins the N- and C-termini of OmpX.
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|-
-
 
+
|'''ehaB'''
-
CPG_L2 - circularly permuted OmpG(organism Escherichia Coli)
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||Organism: Escherichia coli<br>
-
This protein is circularly permuted with its backbone opening in loop 2.  
+
Features: primary sequence alone is sufficient for crossing the bacterial membrane
-
 
+
|-
-
CPG_L6 - circularly permuted OmpG(organism Escherichia Coli)
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|'''TshA'''
-
This protein is circularly permuted with its backbone opening in loop 6.
+
||Organism: Escherichia coli<br>
-
 
+
Autotransporter type: serine protease subfamily (because of the 7AA serine protease motif)<br>
-
 
+
|-
-
 
+
| '''VirG(IcsA)'''
-
Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test the functionality of these putative autotransporters and expand the range of displayers available for surface display.
+
||Organism: Shigella flexneri
 +
|-
 +
|'''YuaQ AtD (putative)'''
 +
||Organism: Escherichia coli<br>
 +
Features: bears sequence similarity to the confirmed autotransporters AIDA and Ag43
 +
|-
 +
|'''AIDA-I'''
 +
||Organism: Escherichia Coli<br>
 +
Features: identified to be similar to IgA1, the first autotransporter used for surface display. Occurs naturally in the host organism, E. coli, and is a robust tool for surface display
 +
|-
 +
|'''Ag43_short'''
 +
||Organism: Escherichia Coli MG1655<br>
 +
Features: expression of Ag43 is evenly distributed around the bacterial cell<br>
 +
Structure: 14 antiparallel beta strands each composed of about 12AA residues
 +
|-
 +
|'''eCPX (circularly permuted OmpX)'''
 +
||Organism: Escherichia Coli<br>
 +
Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX.<br>
 +
[[Image:Ecpx image using molecular operating environment.jpg|120px]]
 +
|-
 +
|'''CPG_L2 (circularly permuted OmpG)'''
 +
||Organism: Escherichia Coli<br>
 +
Features: protein is circularly permuted with its backbone opening in loop 2, allowing both the N- and C- termini to be present in the extracellular space.
 +
|-
 +
|'''CPG_L6 (circularly permuted OmpG)'''
 +
||Organism: Escherichia Coli<br>
 +
Features: protein is circularly permuted with its backbone opening in loop 6, allowing both the N- and C- termini to be present in the extracellular space.
 +
|}
 +
Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test their functionality and expand the range of displayers available for surface display.
==References==
==References==
Pina, S et al. Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. J Bacteriol. January 2009; 191(2): 576–587. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).  
Pina, S et al. Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. J Bacteriol. January 2009; 191(2): 576–587. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).  
 +
 +
Kostakioti, M et al. Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain. Infect Immun. October 2004;72(10):5548-54. Available Online: http://www.ncbi.nlm.nih.gov/pubmed/15385451?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum (Accessed: 20 October 2009).
http://www.uniprot.org/uniprot/?query=H81868&sort=score
http://www.uniprot.org/uniprot/?query=H81868&sort=score
Line 155: Line 163:
http://www.genome.jp/dbget-bin/www_bget?pcr:Pcryo_1225
http://www.genome.jp/dbget-bin/www_bget?pcr:Pcryo_1225
 +
 +
http://www.uniprot.org/uniprot/Q9JMS3

Latest revision as of 02:17, 22 October 2009



Cell Surface Display Parts


Schematic.jpg

Passengers

Follow any of the links below to see assay information for each of the passengers we made.

[http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_MGFP http://2009.igem.org/wiki/images/6/63/Mgfp5button.png]

MGFP-5
The Sticky Protein
Displaying this sticky peptide would allow you to make an underwater bio-glue!

[http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Cellulases http://2009.igem.org/wiki/images/d/df/Cellulasebutton.png]

Cellulase
The Enzyme
Displaying enzymes that degrade cellulose would allow consolidated bioprocessing for biofuel production!

 [http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_TypeIII_Needle_scFv http://2009.igem.org/wiki/images/d/d8/Scfvbutton.png]

Type III Needle scFv
The Antibody Fragment
Displaying antibody fragments would allow easy pathogen detection!

[http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Streptavidin http://2009.igem.org/wiki/images/a/aa/Ag4.png]

Streptavidin Binding Tag
The Tag
A tag on the surface of E.coli would allow you to purify bacteria preferentially!

 [http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Leucine_Zippers http://2009.igem.org/wiki/images/d/df/Leucinezipeprsbutton.png]

Leucine Zippers
The Heterodimeric Binding Peptide
Displaying heterodimeric binding peptides would allow you to make heterogeneous bacteria systems!

 [http://2009.igem.org/Team:Berkeley_Wetlab/Passenger:_Ag4_Peptide http://2009.igem.org/wiki/images/2/22/Streptagbutton.png]

Ag4
The Silver Binding Peptide
The metal head of our set! Displaying a heavy metal binding peptide would promote bioremediation of heavy metals and aid in synthesis of biomaterials.

Spacers

Spacer elements are present in most natural display systems, which suggests that they are important components of cell surface display. However, the precise role of spacers in the functionality of outer membrane proteins has not been extensively characterized and most people engineer cell surface display devices without structural spacers. We decided to build a few cell surface display systems with spacer elements in order to begin characterizing their role within engineered cell surface display devices. We introduced five different spacer domains to our surface display system: INP-repeats, beta roll, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below.

INP-Repeats Synthetic spacer region composed of repeated portions of ice nucleation protein (INP) sequence.
Beta Roll Protein tertiary structure that is natively found in autotransporters and other outermembrane proteins.
Beta Helix Protein helical structures stabilized by hydrogen bonds and protein-protein interactions. The resulting structure contains two to three faces formed by the association of parallel beta strands.
Gly-Ser Repeats Synthetic spacer made from fourteen repeated glycine and serine residues. This spacer is essentially devoid of secondary structure, it is simply a long flexible linker. We made in order to differentiate between the importance of a natural spacer with secondary/tertiary structure and a long linker between the displayer and its passenger.
GFP-LVA Modified green fluorescent protein

Displayers

A displayer is defined as an outmembrane protein that carries another protein to the extracellular space of the cell.

For successful cell surface display of proteins, there must be an effective protein localization mechanism. Gram-negative bacteria such as E. Coli have two membranes, which present a problem for transporting proteins synthesized in the cytoplasm to the outside of the cell. Various transport schemes exist in gram-negative bacteria to effectively localize proteins to the outermembrane. The most common schemes are TypeI, TypeIII, and TypeV secretion.

In our display systems, we chose a class of outermembrane proteins called autotransporters that localizes proteins via the TypeV secretion mechanism. Over 700 autotransporters have been sequenced, many of which are used to export virulence factors to the outside of the cell. We decided to harvest this localization system for cell surface display because the outermembrane protein (aka displayer) spontaneously inserts into the outermembrane and pulls the protein it is covalently linked to (aka passenger) into the extracellular space. Moreover, autotransporters are capable of pulling through large proteins, such as enzymes and single-chain variable fragments. We have removed the native passengers in our autotransporter constructs and fused heterologous passengers of interest to the N-terminus of these autotransporters.

Autotransporter secretion.png

As depicted in the diagram above, autotransporter transport begins with localization to the periplasm via the Sec secretion pathway. The translocated protein remains unfolded in the periplasm until it inserts into the outermembrane by forming a beta barrel with its C-terminal 250-300 amino acyl residues. The N-terminus of the protein (containing our passenger of interest) is then pulled through the barrel to the outside of the cell. Passengers of displayers are often cleaved for extracellular secretion. In our systems, however, we removed the signal sequence that signals for peptide cleavage so our passengers remain attached to the transmembrane displayer protein.

In constructing our parts, we looked into a broad range of autotransporters, some well characterized and others putative, to explore the spectrum of display machinery and to establish the functionality of novel autotransporters for cell surface display.

Azo1653 AtD (putative) Organism: Azoarcus sp. (strain BH72)

Autotransporter type: AT-1 family

OprF AtD Organism: Pseudomonas fluorescens

Structure: an 8-stranded beta barrel in the outermembrane

Cl02365 AtD (putative) Organism: Neisseria meningitidis

Autotransporter type: AT-1 family

VtaA11 AtD Organism: Haemophilus parasuis

Autotransporter type: AT-2 family

Hag AtD Organism: Moraxella catarrhalis

Autotransporter type: dimeric family
Structure: 200kDa protein with 10-stranded beta barrel
predicted 2D structure of Hag AtD

Pcryo_1225 AtD (putative) Organism: Psychrobacter cryohalolentis
Hia AtD Organism: Haemophilus influenzae

Autotransporter domain: trimeric family
Structure: modular segments containing repeats of structurally distinct domains
Hia ATD.jpg

upaG_short Organism: Escherichia Coli

Autotransporter type: trimeric family

espP(beta) Organism: Escherichia coli

Structure: 12-stranded beta barrel
EspP ATD.jpg

ehaB Organism: Escherichia coli

Features: primary sequence alone is sufficient for crossing the bacterial membrane

TshA Organism: Escherichia coli

Autotransporter type: serine protease subfamily (because of the 7AA serine protease motif)

VirG(IcsA) Organism: Shigella flexneri
YuaQ AtD (putative) Organism: Escherichia coli

Features: bears sequence similarity to the confirmed autotransporters AIDA and Ag43

AIDA-I Organism: Escherichia Coli

Features: identified to be similar to IgA1, the first autotransporter used for surface display. Occurs naturally in the host organism, E. coli, and is a robust tool for surface display

Ag43_short Organism: Escherichia Coli MG1655

Features: expression of Ag43 is evenly distributed around the bacterial cell
Structure: 14 antiparallel beta strands each composed of about 12AA residues

eCPX (circularly permuted OmpX) Organism: Escherichia Coli

Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX.
Ecpx image using molecular operating environment.jpg

CPG_L2 (circularly permuted OmpG) Organism: Escherichia Coli

Features: protein is circularly permuted with its backbone opening in loop 2, allowing both the N- and C- termini to be present in the extracellular space.

CPG_L6 (circularly permuted OmpG) Organism: Escherichia Coli

Features: protein is circularly permuted with its backbone opening in loop 6, allowing both the N- and C- termini to be present in the extracellular space.

Some of these proteins are putative autotransporters that have sequence homology to confirmed autotransporters. We chose these proteins because we wanted to test their functionality and expand the range of displayers available for surface display.

References

Pina, S et al. Trimeric Autotransporters of Haemophilus parasuis: Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. J Bacteriol. January 2009; 191(2): 576–587. Available Online: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620822/ (Accessed: 20 October 2009).

Kostakioti, M et al. Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain. Infect Immun. October 2004;72(10):5548-54. Available Online: http://www.ncbi.nlm.nih.gov/pubmed/15385451?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum (Accessed: 20 October 2009).

http://www.uniprot.org/uniprot/?query=H81868&sort=score

http://pfam.sanger.ac.uk/family?acc=PF05736

http://www.genome.jp/dbget-bin/www_bget?azo:azo1653

http://www.genome.jp/dbget-bin/www_bget?pcr:Pcryo_1225

http://www.uniprot.org/uniprot/Q9JMS3

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