Cell Surface Targeting: An Update'

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Cell Surface TargetingAn Update.

• 7/10/2006

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Outline

• Streptavidin Fusion protein
• OmpA
• Other scaffolds
• Adaptamers

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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
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Progress

• Streptavidin clones (WT, WTHis6, Dead) obtained
  from M. Howarth working to get SCD clone from F.
  Aslan
• Georgiou lab unresponsive with Lpp-OmpA
• Primers ordered to PCR Howarths clones and full
  OmpA from K12 genome
• Will order primers to PCR Aslans clone and to
  PCR OmpA 46-159/46-66/synthesize Lpp tag?

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Other scaffolds (GPI-anchors, Autotransporter)

• No one else got back to us!
• Autotransporter
• Couldnt get strains genomic DNA
• Another strain available, but autotransporter
  differs greatly from desired construct

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Adaptamers

• Experiment 1 Can we create a simple linker
• Tahiri-Alaouis CopA/CopT construct
• Will try different lengths of linker regions.

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Streptavidin/Thrombin Adaptamer

• Vary linker sizes
• Gel shift experiments to determine if any of
  these constructs are able to bind both thrombin
  and streptavidin

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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.