Team:Berkeley Wetlab/Cell Surface Display Parts

Cell Surface Display Parts

Follow any of the links below for more information about 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!

• 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 inner and outer membranes that 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. This system is particular suited 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, 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-terminus. The N-terminus of the protein (containing our passenger of interest) is then pulled through the barrel to the outside of the cell.

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

azo1653 AtD

OprF AtD Cl02365 AtD VtaA11 Hag AtD Pcryo_1225AtD

Hia AtD - species Haemophilus influenzae

upaG_short

espP(beta) - species Escherichia Coli

ehaB TshA VirG YuaQ AtD AIDA-I AtD Ag43_short

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

Their precise role in the functionality of proteins has not been extensively characterized.

Repeated portion of ice nucleation protein (INP) sequence.

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