Cell Surface Targeting
description
Transcript of 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
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG EcoRI XbaI
TACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI
Standard part
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA EcoRI XbaI
ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI
Protein domain
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA EcoRI XbaI
ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI
Domain assembly
ACTAGAThrArg Mixed
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Streptavidin
• Found in bacteria Streptomyces avidinii• Full-length ~160 aa’s, core ~ 140 aa’s• 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.