Presentation on theme: "Streptavidin McDevitt, 1999 It binds to biotin.. Streptavidinfo Found in bacteria Streptomyces avidinii Full-length ~160 aa ’ s, core ~ 140 aa ’ s Binds."— Presentation transcript:
Streptavidinfo Found in bacteria Streptomyces avidinii Full-length ~160 aa ’ s, core ~ 140 aa ’ s Binds to biotin (vitamin H or B7) K d ~ 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 G113- W120 loop to increase solubility Qureshi, 2001: S45A, T90A, D128A to form soluble, functional monomers, K d = 1.7x10 -6 M Qureshi, 2002: T90A, D128A, K d = 1.3x10 -8 Wu, 2005: T76R, V125R (monomer) V55T, L109T (soluble), reported better solubility than Qureshi 2002, K d = 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/constructs1 wk PCR into BioBricks; mutagenesis; sequencing. 3 wks Assembly and cloning.2 wks Test functionality.2 wks 3-4 people.