Streptavidin

Streptavidin (pronounced /ˌstrɛpˈtævədən/) is a 52,800 dalton^[1] tetrameric protein purified from the bacterium Streptomyces avidinii. It finds wide use in molecular biology through its extraordinarily strong affinity for biotin (also known as vitamin H); the dissociation constant (K_d) of the biotin-streptavidin complex is on the order of ~10^-15 mol/L, ranking among the strongest non-covalent interactions known in nature.

Structure

The crystal structure of streptavidin with biotin bound was first solved in 1989 by Hendrickson et al. ^[2] and as of May 2009, there are 134 structures deposited on the RCSB Protein Data Bank. The N and C termini of the 159 residue full-length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13 - 139; heterogeneous products can result when the termini are processed differently. The secondary structure of a streptavidin monomer is composed of eight antiparallel β-strands, which fold to give an antiparallel beta barrel tertiary structure. A biotin binding-site is located at one end of each β-barrel, which has a high affinity as well as a high avidity for biotin. Four identical streptavidin monomers (i.e. four identical β-barrels) associate to give streptavidin’s tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers.

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. A further application is the so called Strep-tag, which is an optimized system for the purification and detection of proteins.

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.

Pretargetted Immunotherapy
This uses streptavidin conjugated to a monoclonal antibody against cancer cell-specific antigens followed by an injection of radiolabelled biotin, to deliver the radiation only to the cancerous cell. Initial hurdles involve saturation of the biotin binding sites on streptavidin with endogenous biotin instead of the injected radiolabelled biotin, and a high degree of radioactive exposure in the kidneys, due to streptavidin’s strong cell adsorptive properties. It is currently thought that this high level of binding to adherent cell types, such as activated platelets and melanomas, is a result of integrin binding mediated through the RYD sequence in streptavidin^[3].

Monovalent and monomeric streptavidin

Streptavidin is a tetramer and each subunit binds biotin with equal affinity. Multivalency is an advantage in some applications, for example where avidity effects improve the ability of molecules attached to streptavidin to detect specific T cells ^[4]. In other cases, such as the use of streptavidin for imaging specific proteins on cells, multivalency can perturb the function of the protein of interest. Monovalent streptavidin is an engineered recombinant form of streptavidin which is a tetramer but only one of the four binding sites is functional^[5]. This single binding site has 10^-14 mol/L affinity and cannot cause cross-linking.

Monomeric streptavidin is a recombinant form of streptavidin with mutations to break the tetramer into a monomer and to enhance the solubility of the resultant isolated subunit. Monomeric streptavidin has an affinity for biotin of 10^-7mol/L and so is not ideal for labeling applications but is good for purification, where reversibility is desirable^[6].

Comparison to avidin

Streptavidin is not the only protein capable of binding to biotin with high affinity. Avidin is the other most notable biotin-binding protein, which is evolutionarily unrelated to streptavidin but has very similar properties. Originally isolated from egg white, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. It has a higher affinity for biotin (Kd ~ 10^-15M) but in contrast to streptavidin, it is glycosylated, positively charged, has pseudo-catalytic activity (it can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation.

Streptavidin has a mildly acidic isoelectric point (pI) of ~5, but a recombinant form of streptavidin with 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.

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:10.1093/nar/22.3.492. PMID 8127690. 

External links

• Swiss-Prot entry for Streptavidin precursor from Streptomyces avidinii
• MeSH Streptavidin