Team:Berkeley Wetlab/Cell Surface Display Parts

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Cell Surface Display Parts


Schematic.jpg

Contents

Passengers

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


Streptavidin
A tag that binds the protein streptavidin!


Leucine Zippers
A structural motif that can allow different cell types to recognize and to bind each other!


Ag4 Peptide
A peptide that reduces silver ions to form a silver precipitate!


MGFP-5
A protein used by mussels to stick to rocks. An underwater bio-glue!


Cellulases
Enzymes that degrade cellulose!


TypeIII Needle scFv
An antibody that binds a motif common to enteropathogenic bacteria!

Spacers

Spacer elements occur in natural membrane protein systems. This is exemplified in the Hag protein, autotransporter-containing system containing two spacer beta roll domains, shown below:


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The precise role of spacers in the functionality of proteins has not been extensively characterized; however, as illustrated in the Hag protein, multiple spacer elements are typically present in natural display systems suggesting their importance. 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.


Inp-repeats.png
Repeated portion of ice nucleation protein (INP) sequence.

Betaroll.png


Beta helix pic.jpg
Beta helices are 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.png

Gfp-lva.png

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.

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.

Autotransporter secretion.png

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.

In constructing our parts, we looked into various autotransporters with different attributes conducive to cell surface display.

azo1653 AtD (putative) - organism Azoarcus sp. (strain BH72)
This protein contains one autotransporter domain of the AT-1 family.

OprF AtD - organism Pseudomonas fluorescens

Cl02365 AtD - organism Neisseria meningitidis

VtaA11 AtD - organism Haemophilus parasuis

Hag AtD - organism Moraxella catarrhalis

Pcryo_1225AtD - organism Psychrobacter cryohalolentis

Hia AtD - organism Haemophilus influenzae Hia ATD.jpg

upaG_short - organism Escherichia Coli

espP(beta) - organism Escherichia coli EspP ATD.jpg

ehaB - organism Escherichia coli

TshA - organism Escherichia coli
subgroup1 of autotransporters

VirG(IcsA) - organism Shigella flexneri

YuaQ AtD - organism Escherichia coli

AIDA-I - organism Escherichia Coli

Ag43_short - organism Escherichia Coli MG1655

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.

References