Streptavidin
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Streptavidin is a 53,000 dalton[1] tetrameric protein purified from the bacterium Streptomyces avidinii. It finds wide use in molecular biology through its extraordinarily strong affinity for the vitamin biotin; the dissociation constant (Kd) of the biotin-streptavidin complex is on the order of ~10-15 mol/L, ranking among one of the strongest known non-covalent interactions.
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[edit] Uses in Biotechnology
Among the most common uses are the purification or detection of various biomolecules. The strong streptavidin-biotin bond can be used to attach various biomolecules to one another or onto a solid support.
One technique fixes DNA by first digesting DNA with a restriction exonuclease to produce either a blunt end, a 3' overhang or a 5' overhang. The DNA is then incubated with biotin-11-dUTP, a deoxyribonucleotide analog that is covalently attached to biotin, and the Klenow fragment of the holoenzyme DNA polymerase I of E. coli. The biotin-11-dUTP is incorporated into the 3' end of the strand complementary to the 5' ssDNA portion of the overhang. This is because DNA polymerases can only add nucleotides to the 3'-OH of DNA and not the 5'-phosphate.
- 5'-ACTGGCTU-3'
- 3'-TGACCGAACCGTT-5'
(where U is biotin-11-UTP incorporated into the DNA strand)
Assuming that care is taken to ensure that only one 5' overhang with only one possible site is available for dUTP incorporation, the result is a strand of DNA with a biotinylated end. One of the primary uses for biotinylated DNA is for binding (via non-covalent interactions) to streptavidin coated surfaces. With the DNA firmly attached to this substrate, various DNA hybridization and immunological assays can be performed. They may also be attached to streptavidin coated agarose microspheres, polystyrene or even paramagnetic beads. These complexes are most commonly used for the purification or isolation of DNA binding proteins. Other molecular biological techniques allow for exquisite control over DNA sequence, length, etc which make this a very powerful molecular tool.
[edit] Comparison to avidin
There are considerable differences in the composition of avidin and streptavidin, but they are remarkably similar in other respects. Both proteins form tetrameric complexes to function in which each subunit can bind one molecule of biotin. Guanidine hydrochloride will dissociate both avidin and streptavidin tetramers into their component subunits, but streptavidin is more resistant to dissociation.
Streptavidin is much less soluble in water than avidin, and it lacks avidin's extensive glycosylation. Streptavidin has a mildly acidic isoelectric point (pI) of ~5. A recombinant form of streptavidin with a mass of 53,000 daltons and a near-neutral pI is also commercially available. Because streptavidin lacks any carbohydrate modification and has a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. Deglycosylated avidin is more comparable to the size, pI and nonspecific binding of streptavidin.
[edit] References
- Wilchek, M. and Bayer, E.A. (1989). Protein Recognition of Immobilized Ligands. Hutchins, T.W., ed. Alan R. Liss, Inc., pp. 83-90.
- Current Protocols in Protein Science (1998) 9.7-9.7.13
- Zimmermann R, Cox E (1994). "DNA stretching on functionalized gold surfaces". Nucleic Acids Res. 22 (3): 492-7. doi:. PMID 8127690.
