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Streptavidin McDevitt, 1999 It binds to biotin.. Streptavidinfo Found in bacteria Streptomyces avidinii Full-length ~160 aa ’ s, core ~ 140 aa ’ s Binds.

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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:

1 Streptavidin McDevitt, 1999 It binds to biotin.

2 Streptavidinfo Found in bacteria Streptomyces avidinii Full-length ~160 aa ’ s, core ~ 140 aa ’ s Binds to biotin (vitamin H or B7) K d ~ 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, 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

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/constructs1 wk PCR into BioBricks; mutagenesis; sequencing. 3 wks Assembly and cloning.2 wks Test functionality.2 wks 3-4 people.


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