Cell Surface Targeting:An Update.

7/10/2006

OutlineStreptavidin 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 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

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?

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

Adaptamers Experiment 1: Can we create a simple linker

Tahiri-Alaoui’s CopA/CopT construct

Will try different lengths of linker regions.

Streptavidin/Thrombin Adaptamer Vary linker sizes Gel shift

experiments to determine if any of these constructs are able to bind both thrombin and streptavidin

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.