Team:Berkeley Wetlab/Cell Surface Display Parts

Cell Surface Display Parts

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

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.

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.



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.

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.