Coding

Part:BBa_K197015

Designed by: Gabriela Guzman Lopez Aguado   Group: iGEM09_Berkeley_Wetlab   (2009-10-21)

{eCPX (circularly permuted OmpX)}



This part is a cell-surface displayer part. 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. The diagram below describes the outcome of displaying a panel of passenger proteins using this set of displayers. Below it is a full list of display systems included in the set.

HeatMapBerkeley.jpg

The heat map above points to an interesting trend made clear by the streptavidin and mgfp-5 data. Although all constructs contain short linkers between the displayers and passengers, the inclusion of spacer elements for both systems appears to enhance functional surface display of the passengers. Moreover, the identity of the spacer element is an important parameter determining display efficiency. There is an increase in functional display when the INP repeats spacer is added between the displayers and the strep tag as is seen in the increase in lighter blocks in the map. This trend is especially evident in the mgfp data in which there are several weak signals (many dark blocks in the map) for mgfp displayed on its own. With the addition of several spacer elements, a significant general increase in signal for almost 100% of the systems is observed.

Many of our display systems are derived from type V secreted, or autotransporter domain-containing proteins. 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

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

upaG_short Organism: Escherichia Coli

Autotransporter type: trimeric family

espP(beta) Organism: Escherichia coli

Structure: 12-stranded beta barrel

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.

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).


Features: protein is an enhanced CPX variant located in the outermembrane that joins the N- and C-termini of OmpX. Autotransporters are proteins that form a pore in the outermembrane and and pull their N terminus through this pore in order to expose it to the extracellular space. N-terminal fusions of proteins to this part may permit the cell surface display of the fused protein.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 189
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 189
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 189
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 189
  • 1000
    COMPATIBLE WITH RFC[1000]


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