Transcript Streptavidin - OpenWetWare

Streptavidin
It binds to biotin.
McDevitt, 1999
Streptavidinfo
Found in bacteria Streptomyces avidinii
Full-length ~160 aa’s, core ~ 140 aa’s
Binds to biotin (vitamin H or B7)

Kd ~ 10-15 M (Chaiet, 1964)
Forms tetramers



Strong monomer-monomer interactions
Weak dimer-dimer interactions (Sano, 1997)
Biotin binding relies heavily (10-7) on Trp-120 of
neighboring subunit (Sano, 1995)
No cysteines, no carbohydrates, no charge,
no problem
The goal
To create a fusion protein in E. coli
expressing streptavidin on the surface

Can bind anything biotinylated
Peptides
 DNA
 Antibodies

Problems
Toxicity
Tetramer vs. monomer
Solubility
Toxicity
Biotin is important for many of E.coli’s metabolic
pathways.
Streptavidin binds biotin and makes it unavailable.
Solution: T7 promoter/RNA polymerase
This was for one-time expression/harvesting (~35%
of total protein in Sano, 1990).
Engineering by mutation
Introduce amino acid mutations to disrupt tetramer
formation and to improve solubility
Sano, 1997: H127D to form dimers; delete G113W120 loop to increase solubility
Qureshi, 2001: S45A, T90A, D128A to form soluble,
functional monomers, Kd = 1.7x10-6 M
Qureshi, 2002: T90A, D128A, Kd = 1.3x10-8
Wu, 2005: T76R, V125R (monomer) V55T, L109T
(soluble), reported better solubility than Qureshi
2002, Kd = 2.2x10-7
Looser biotin affinity
Reversibility
Purification
 Recyclable

Non-toxicity (Wu, 2006)
Still good binding
Concerns for this
project
Toxicity may not be an issue when
expressed on the surface.
We don’t “need” monomers.
We do need solubility ~ hydrophilicity.
In other news
A bifunctional chimeric protein

Streptavidin + MMP inhibitor (Farlow, 2002)
Cell recognition peptides

RGD adhesion sequence to rat aortic endothelial
and human melanoma cells (McDevitt, 1999)
Streptavidin-based containment systems

99.9% culture suicide in 8 hr, induced by absence
of hydrocarbon substrate (Kaplan, 1999)
Agenda
Design and order primers, obtain streptavidin and
membrane protein genes/constructs
1 wk
PCR into BioBricks; mutagenesis; sequencing.
3 wks
Assembly and cloning.
2 wks
Test functionality.
2 wks
3-4 people.