Team:Berkeley Wetlab/Cell Surface Display Parts

Passengers

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

The Tag A tag that binds the protein streptavidin!	The Heterodimeric Binding Peptide A structural motif that can allow different cell types to recognize and to bind each other!	The Other Binding Peptide A peptide that reduces silver ions to form a silver precipitate!
The Sticky Protein A protein used by mussels to stick to rocks. An underwater bio-glue!	The Enzyme Enzymes that degrade cellulose!	The scFv An antibody fragment that binds a motif common to enteropathogenic bacteria!

Spacers

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, beta helix, Gly-Ser repeats and GFP-LVA. These elements are further described below.

INP - Repeats-repeated portion 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 - a flexible 14-mer amino acid sequence of glycine and serine residues repeated.

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.

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.

1. azo1653 AtD (putative) - organism Azoarcus sp. (strain BH72)
Autotransporter domain: AT-1 family

2. OprF AtD - organism Pseudomonas fluorescens
Structure: an 8-stranded beta barrel in the outermembrane

3. Cl02365 AtD (putative) - organism Neisseria meningitidis
Autotransporter domain: AT-1 family

4. VtaA11 AtD - organism Haemophilus parasuis
Autotransporter domain: AT-2 family

5. Hag AtD - organism Moraxella catarrhalis
Autotransporter domain: dimeric family Structure: 200kDa protein with 10-stranded beta barrel

6. Pcryo_1225 AtD (putative) - organism Psychrobacter cryohalolentis

7. Hia AtD - organism Haemophilus influenzae
Autotransporter domain: trimeric family Structure: modular segments containing repeats of structurally distinct domains

8. upaG_short - organism Escherichia Coli
Autotransporter domain: trimeric family

9. espP(beta) - organism Escherichia coli

ehaB - organism Escherichia coli

TshA - organism Escherichia coli
subgroup1 of autotransporters

VirG(IcsA) - organism Shigella flexneri

YuaQ AtD (putative) - organism Escherichia coli
Similarity: bears sequence similarity to the confirmed autotransporters AIDA and Ag43
Size: 1371AA

AIDA-I - organism Escherichia Coli

Ag43_short - organism Escherichia Coli MG1655

eCPX (circularly permuted OmpX)- organism Escherichia Coli
This protein is an enhanced CPX variant that is located in the outermembrane that joins the N- and C-termini of OmpX.

CPG_L2 (circularly permuted OmpG)- organism Escherichia Coli This 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 This protein is circularly permuted with its backbone opening in loop 6.

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

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).

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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