Transcript Cell Surface Targeting

Cell Surface Targeting
Two routes
Protein-protein interface
DNA-DNA interface
Protein-protein
Protein domain BioBricks
Standard part
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG
EcoRI
XbaI
TACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’
SpeI
PstI
Protein domain
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA
EcoRI
XbaI
ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’
SpeI
PstI
Domain assembly
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA
EcoRI
XbaI
ACTAGA
ThrArg
Mixed
ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’
SpeI
PstI
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 aptamerprotein 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.