Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Patent
1998-03-05
1999-10-26
Ceperley, Mary E.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
435 6, 435 75, 435188, 436526, 436527, 436531, 530367, 530405, C07K 1477, G01N 33532
Patent
active
059731249
DESCRIPTION:
BRIEF SUMMARY
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to an avidin-type molecule which is modified at the binding-site tyrosine residue, known to be critical to the binding of biotin. The modified avidin is still capable of binding biotin or a biotinylated ligand under specific conditions, but upon altering these conditions, for example, high pH or competition with biotin, the bound biotin moiety or biotinylated ligand is removed. The invention thus provides a reversible form of avidin for use in avidin-biotin technology, thus "correcting" one of the major disadvantages of the avidin molecule for various applicative purposes, i.e., the extreme denaturing conditions required to disrupt the avidin-biotin complex. These drastic conditions necessary to dissociate the avidin-biotin complex usually inactivate irreversibly the biological activity of the biotinylated component, thus rendering it unsuitable for subsequent use.
Avidin (from egg-white) and streptavidin (from Streptomyces avidinii) are two related proteins that bind biotin with similar dissociation constants of about 10.sup.-15 M (Green, 1975). In addition to the binding of biotin, many of their physical properties are quite similar. Both, for example, are constructed of four non-covalently attached identical subunits, each of which bears a single biotin-binding site. The subunit M.sub.r values are also very similar. Moreover, several short stretches in the sequences of the two proteins are preserved, particularly to Trp-Lys stretches that occur at approximately similar positions (Argarana et al., 1986). We have previously shown (Gitlin et al., 1987, 1988a) that certain lysine and tryptophan residues are involved in the biotin binding in both proteins (Gitlin et al., 1988b). More recently, it was shown that both avidin and streptavidin exhibit the same three dimensional fold, and the most of the binding site residues are identical or similar (Weber et al., 1989). The binding site geometry and bonds formed between both proteins with the biotin molecule are indeed very similar.
Despite these similarities, several differences exist between the two proteins. Avidin is a disulphide-bridged glycoprotein containing two methionine residues, whereas streptavidin is not glycosylated and is devoid of sulphur-containing amino acid side chains. Another significant difference is in the tyrosine content. Avidin has only one tyrosine residue (Tyr-33), whereas streptavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosine residue of a avidin is located in a region which contains a sequence identical with that of one of the streptavidin tyrosine residues (Tyr-43 in the stretch Thr-Gly-Thr-Tyr). This tyrosine residue occupies a prominent position in the biotin-binding site and the chemical modification of the tyrosine hydroxyl group leads to irreversible inactivation of the avidin molecule (Gitlin et. al., 1990).
Each avidin monomer binds one molecule of biotin. The unique feature of this binding, of course, is the strength and specificity of formation of the avidin-biotin complex. The resultant affinity constant, estimated at 1.6.times.10.sup.15 M.sup.-1 for avidin and 2.5.times.10.sup.13 M.sup.-1 for streptavidin (Green, 1990), is the highest known for a protein and an organic ligand. It is so strong that biotin cannot be released from the binding site, even when subjected to a variety of drastic conditions such as high concentrations of denaturing agents at room temperature, e.g., 6M guanidinium hydrochloride, 3M guanidinium thiocyanate, 8M urea, 10% .beta.-mercaptoethanol or 10% sodium dodecyl sulfate. Under combined treatment with guanidinium hydrochloride at low pH (1.5) or upon heating (>70.degree. C.) in the presence of denaturing agents or detergents, the protein is denatured, and biotin is dislodged from the disrupted binding site.
Avidin recognizes biotin mainly at the ureido (urea-like) ring of the molecule. The interaction between the binding site of avidin with the sulfur-containing ring
REFERENCES:
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Bayer Edward A.
Morag Ely
Wilchek Meir
Ceperley Mary E.
Yeda Research and Development Co. Ltd.
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