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Streptavidin

It binds to biotin. Streptavidin. McDevitt, 1999. 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

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Streptavidin

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  1. It binds to biotin. Streptavidin McDevitt, 1999

  2. 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

  3. The goal • To create a fusion protein in E. coli expressing streptavidin on the surface • Can bind anything biotinylated • Peptides • DNA • Antibodies

  4. Problems • Toxicity • Tetramer vs. monomer • Solubility

  5. 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).

  6. 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, 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

  7. Looser biotin affinity • Reversibility • Purification • Recyclable • Non-toxicity (Wu, 2006) • Still good binding

  8. 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.

  9. 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)

  10. 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.

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