Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-03-23
2002-04-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S069100, C530S350000, C530S300000, C536S023700, C536S023100
Reexamination Certificate
active
06368813
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compounds and methods, and in particular, modified streptavidin having affinity for biotin substitutes.
BACKGROUND
The biological sciences have been employed since early times by mankind to modify living organisms or their constitutive elements for a variety of purposes, such as the production of foods and therapeutic agents. However, only during the last fifty years there has been progress at the genetic level to gain a much better understanding of the essential component of living systems. This has led to the understanding that nucleic acids, in the form of DNA and RNA, store and distribute genetic information that determines the sequences of amino acids that characterize proteins; proteins contribute to the structure of an organism and execute most of the tasks required for its function and that even proteins form part of the mechanism by which they are synthesized (e.g. chaperones); polysaccharides, linear and branched polymer of sugars, provide structural elements, store energy, and when combined with peptides or proteins play an important role in cellular recognition; lipids, which include molecules such as fatty acids, phospholipids, and cholesterol, serve as energy sources and are the most important components of the membrane structures that organize and compartmentalize cellular function.
However, proteins are the biological macromolecules with the greatest functional diversity. Proteins catalyze most reactions that occur in living cells, or serve as inhibitors of enzymatic reactions. They transport oxygen, electrons and energy to specific regions in the cell. Other proteins protect living organisms by recognizing and binding to foreign substances. There are also proteins that have a structural function such as collagen, the main constituent of connective tissue fibrils and bones, or have functional roles such as actin or myosin, which are involved in muscle dynamics.
Most proteins biological function is derived from interactions with other molecules such as ligands, hormones, coenzymes or other biological compounds. As a result of this action, there can be important structural changes in both the protein and the other molecule. These conformational changes, in many occasions, are essential for activity; but in other cases they are not as relevant. In hormone-receptor binding, for example, structural changes are fundamental to the transmission of information. Consequently, elucidation of reactive, and non-reactive interactions that are possible between a protein and a ligand is essential for the correct understanding of the molecular mechanisms that govern protein recognition by another molecule.
One of the most remarkable non-reactive protein-ligand interaction involves a 60-kDa tetrameric protein that originates from the actinobacterium
Streptomyces avidinii
termed streptavidin [Chaiet and Wolf, “The Properties of Streptavidin, a biotin-binding protein produced by Streptomycetes,”
Arch. Biochem. Biophys.
106:1-5 (1964)] and the small organic molecule biotin. The binding of biotin by streptavidin is accompanied by one of the largest decreases in free energy observed for a non-covalent interaction in aqueous solution (Ka~1015 M-1)[Green, “Avidin,”
Adv. Protein Chem.
29:85-133 (1975)].
The high association constant of the streptavidin-biotin complex, which is four to six orders of magnitude higher than most antigen-antibody interactions, has many useful applications in the biological sciences. The streptavidin-biotin system has been exploited to devise widely applicable tools in microbiology [Suzuki et al., “Chemiluminescent enzyme-linked immunoassay for reverse transcriptase, illustrated by detection of HIV reverse transcriptase,”
Anal. Biochem.
210:277-28 (1993)], biochemistry [Katz, “Binding to protein targets of peptidic leads discovered by phage display: crystal structures of streptavidin-bound linear and cyclic peptide ligand containing the HPQ sequence,”
Biochem.
34:15421-15429 (1995)] and biotechnology [Bayer and Wilchek, “The use of the avidin-biotin complex as a tool in molecular biology,”
Methods Biochem. Anal.
26:1-45 (1980); Fuccillo, “Application of the Avidin-Biotin Technique in Microbiology,”
Biotechniques
3:494-501 (1985); Buckland, “Strong signals from streptavidin-biotin,”
Nature
320:557-558 (1986)], as well as in the medical sciences, for example, for the localization and separation of antigens [Zaar, “Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study,”
J. Histochem. and Cytochem.
44:1013-1019 (1996)], immunotherapy [Bodey et al., “Immunophenotypically varied cell subpopulations in primary and metastatic human melanomas. Monoclonal antibodies for diagnosis, detection of neoplastic progression and receptor directed immunotherapy,”
Antican. Res.
16:517-531 (1996)], immunoassay development [Heuer et al., “Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins,”
Biochem.
35:9069-9075 (1996)], Hybridization studies [Nilsson et al., “Real-time monitoring of DNA manipulations using biosensor technology,”
Anal. Biochem.
224:400-408 (1995)], tumor localization [Puy et al., “Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues,”
J. Steriod Biochem. and Mol. Biol.
55:197-209 (1995); Sung et al., “Streptavidin distribution in metastatic tumors pretargeted with a biotinylated monoclonal antibody: theoretical and experimental pharmacokinetics,”
Cancer Res.
54:2166-2175 (1994)] and delivery of radionuclides to cancerous cells [van Osdol et al., “A distributed pharmacokinetic model of two-step imaging and treatment protocols: application to streptavidin-conjugated monoclonal antibodies and radiolabeled biotin,”
J. Nucl. Med.
34:1552-1564 (1993); Kalofonos et al., “Imaging of tumor in patients with Indium-111-labeled biotin and streptavidin conjugated antibodies: preliminary communication,”
J. Nucl. Med.
31:1791-1796 (1990); Pimm et al., “Iodine-131 and indium-111 labeled avidin and streptavidin for pretargeted immunoscintigraphy with biotinylated anti-tumor monoclonal antibody,”
Nucl. Med. Commun.
9:931-941 (1988)].
SUMMARY OF THE INVENTION
The present invention relates to compounds and methods, and in particular, modified streptavidin having affinity for biotin substitutes. The compounds and methods of the present invention are particularly useful where levels of endogenous biotin are present in the system, precluding the use of the standard biotin-avidin approach. In addition, it is contemplated that the streptavidin-biotin system can be used as a model to test if the contacts that exist between a protein and a ligand can serve as the starting point to genetically engineer the protein to develop a high specificity for another ligand. Amino acid substitutions are designed to reduce the affinity for the original ligand and obtain a much higher affinity for the substitute molecule. The guiding consideration for re-designing the biotin-binding site of streptavidin was to significantly reduce biotin-binding by minimizing amino acids substitutions in residues making hydrogen bonds with biotin to preserve this contacts for other biotin-like molecules. To test this, the biotin derivatives 2-iminobiotin and diaminobiotin were selected as biotin substitutes (although other substitutes are possible, including compounds that are not biotin derivatives).
In one embodiment, the present invention contemplates a nucleic acid sequence encoding a streptavidin mutant having a higher affinity for a biotin substitute than for biotin. An illustrative streptavidin mutant has a higher affinity for 2-iminobiotin than for biotin. In a specific embodiment, the sequence encodes a streptavidin mutant consisting of amin
Cantor Charles
Reznik Gabriel O.
Sano Takeshi
Smith Cassandra
Vajda Sandor
Carlson Karen Cochrane
Eisenstein Ronald I.
Mitra Rita
The Trustees of Boston University
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