Cell Surface Targeting

Cell Surface Targeting

Two routes

Protein-protein interface DNA-DNA interface

Protein-protein

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

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

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

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

DNA-DNA

Aptamers

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

• Low generation time

• 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

First designs

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.