Streptavidin

1
Streptavidin
It binds to biotin.
McDevitt, 1999
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Streptavidinfo

• Found in bacteria Streptomyces avidinii
• Full-length 160 aas, core 140 aas
• 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

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The goal

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

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Problems

• Toxicity
• Tetramer vs. monomer
• Solubility

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Toxicity

• Biotin is important for many of E.colis
  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).

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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

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Looser biotin affinity

• Reversibility
• Purification
• Recyclable
• Non-toxicity (Wu, 2006)
• Still good binding

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Concerns for this project

• Toxicity may not be an issue when expressed on
  the surface.
• We dont need monomers.
• We do need solubility hydrophilicity.

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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)

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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.