Cell Surface Targeting: An Update.
description
Transcript of Cell Surface Targeting: An Update.
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.