Streptavidin It binds to biotin Mc Devitt 1999
Streptavidin It binds to biotin. Mc. Devitt, 1999
Streptavidinfo Found in bacteria Streptomyces avidinii Full-length ~160 aa’s, core ~ 140 aa’s Binds to biotin (vitamin H or B 7) l Kd ~ 10 -15 M (Chaiet, 1964) Forms tetramers l l l 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 l Can bind anything biotinylated Peptides l DNA l Antibodies l
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: T 7 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: H 127 D to form dimers; delete G 113 W 120 loop to increase solubility Qureshi, 2001: S 45 A, T 90 A, D 128 A to form soluble, functional monomers, Kd = 1. 7 x 10 -6 M Qureshi, 2002: T 90 A, D 128 A, Kd = 1. 3 x 10 -8 Wu, 2005: T 76 R, V 125 R (monomer) V 55 T, L 109 T (soluble), reported better solubility than Qureshi 2002, Kd = 2. 2 x 10 -7
Looser biotin affinity Reversibility Purification l Recyclable l 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 l Streptavidin + MMP inhibitor (Farlow, 2002) Cell recognition peptides l RGD adhesion sequence to rat aortic endothelial and human melanoma cells (Mc. Devitt, 1999) Streptavidin-based containment systems l 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 Bio. Bricks; mutagenesis; sequencing. 3 wks Assembly and cloning. 2 wks Test functionality. 2 wks 3 -4 people.
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