Cell Surface Targeting

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Cell Surface Targeting
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Two routes
Protein-protein interface
DNA-DNA interface
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Protein-protein
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Protein domain BioBricks
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Streptavidin

• Found in bacteria Streptomyces avidinii
• Full-length 160 aas, core 140 aas
• Binds strongly to biotin (vitamin H or B7)
• Kd 10-15 M (Chaiet, 1964)
• No cysteines, no carbohydrates, no charge

McDevitt, 1999
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Lpp-OmpA surface expression

• 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 stable surface xpression
• 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)
• Goal Use Lpp-OmpA to express streptavidin on
  the surface to bind biotinylated DNA or
  protein to the membrane

Francisco et al., 1992
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Progress/agenda

• Created BioBricks of three streptavidin clones
  from Ting lab wild-type, wild-type His6 tag,
  dead mutant
• Created BioBricks of full Lpp and OmpA, then
  Lpp(1-29), OmpA(46-66), OmpA(46-159)
• More BioBricks single-chain dimer streptavidin
  from Aslan lab
• Assembly and expression of Lpp-OmpA-streptavidin

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DNA-DNA
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Aptamers

• Short DNA/RNA sequences that have high
  specificity and affinity for substrate
• Low generation time

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• Pros
• Potential control of binding kinetics and
  thermodynamics
• Control of relative amounts of different
  substrates that bind cell surface
• Swappable
• Cons
• Interaction strength limited by strength of
  aptamer-protein interactions

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First designs
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Progress

• Have begun to characterize DNA-DNA interaction
• Gel shift assays have failed to prove that
  aptamers are binding the proteins
• For the future more promising assays.