Team:TU Eindhoven/Background/Membrane Anchors

Membrane Anchors: OmpX and Penn Anchor

To be able to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our SPAAC Reaction Page for detailed information.

Membrane Anchor Protein CPX

OmpX (Outer Membrane Protein X) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in E. coli. CPX (Circularly Permuted OmpX) was developed as a bacterial display methodology for N- and C-terminal display. It demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming a semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together by a GGSG-linker, and the newly formed N- and C-termini reside on the cell surface. This makes insertion of an unnatural amino acid fairly easy as it can be positioned before the N-terminus or after the C-terminus. By doing this, the protein itself does not have to be modified. See Figure 1 for a schematic overview of CPX and OmpX.

Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.

Membrane Anchor Protein INPNC

In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: BBa_K811003. INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties. However, INP consists of an N-terminal region that is exposed to the outside membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface display properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed at the cell surface, while the N-terminal domain remains in the outer membrane. Check also the Wiki Page of Penn iGEM Team 2012.