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Cell Surface Targeting: An Update.

Cell Surface Targeting: An Update. 7/10/2006. Outline. Streptavidin Fusion protein OmpA Other scaffolds Adaptamers. Lpp-OmpA surface display vehicle. Lipoprotein, Lpp: major outer membrane protein (most abundant protein in E. coli by numbers); targeted to outer membrane

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Cell Surface Targeting: An Update.

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  1. Cell Surface Targeting:An Update. 7/10/2006

  2. Outline • Streptavidin Fusion protein • OmpA • Other scaffolds • Adaptamers

  3. Lpp-OmpA surface display vehicle • Lipoprotein, Lpp: major outer membrane protein (most abundant protein in E. coli by numbers); targeted to outer membrane • Outer membrane protein A, OmpA: well-characterized eight-strand beta-barrel transmembrane domain • Lpp 20aa signal peptide + Lpp aa 1-9 + OmpA aa 46-159 + surface protein • OmpA 46-66 also successful • Bla, Fv fragments, OPH, Cex (C. fimi) Francisco et al., 1992

  4. Progress • Streptavidin clones (WT, WT+His6, Dead) obtained from M. Howarth; working to get SCD clone from F. Aslan • Georgiou lab unresponsive with Lpp-OmpA • Primers ordered to PCR Howarth’s clones and full OmpA from K12 genome • Will order primers to PCR Aslan’s clone and to PCR OmpA 46-159/46-66/synthesize Lpp tag?

  5. Other scaffolds (GPI-anchors, Autotransporter) • No one else got back to us! • Autotransporter • Couldn’t get strain’s genomic DNA • Another strain available, but autotransporter differs greatly from desired construct

  6. Adaptamers • Experiment 1: Can we create a simple linker • Tahiri-Alaoui’s CopA/CopT construct • Will try different lengths of linker regions.

  7. Streptavidin/Thrombin Adaptamer • Vary linker sizes • Gel shift experiments to determine if any of these constructs are able to bind both thrombin and streptavidin

  8. Future adaptamer work • If any capable of binding… • Change linker sequence, investigate changes in binding affinity • Try out other aptamers/substrates; investigate changes in binding affinity • If not.. • Design linkers with more interesting secondary structures. • Additionally.. • Will continue to bug Dwarakanath about cell surface-binding aptamer • Alternatively, order different cell lines for which surface-binding aptamers have been better documented.

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