Circularly permuted biotin binding proteins

Details Circularly permuted biotin binding proteins Circularly permuted biotin binding proteins

1999-04-02

2002-12-10

Wortman, Donna C. (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C536S023100, C536S023400, C536S023700, C435S069100, C530S825000

Reexamination Certificate

active

06492492

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of modified streptavidin, and more particularly in the area of streptavidin fusion proteins wherein the base polypeptide is streptavidin having a circularly permuted loop.
BACKGROUND OF THE INVENTION
Fusion proteins are polypeptide chains consisting of two or more polypeptides fused together into a single polypeptide chain. Streptavidin fusion proteins have been reported, for example, that combine the biotin binding capabilities of streptavidin with that of a second protein, such as IgG-binding protein A (Sano and Cantor, Bio/Technology 9:1377-1381 (1992), and U.S. Pat. No. 5,328,985 to Sano et al.), metallothionein (Sano, et al., P. N. A. S. USA 89:1534-1538 (1992)), single chain antibodies (Dubel et al., J. Immul. Methods. 178:201-209 (1995)) and the human low density lipoprotein (LDL) receptor (U.S. Pat. No. 4,839,293 to Cantor et al.). These proteins include wild-type streptavidin attached to the second protein. Tight binding of biotin to the streptavidin is substantially maintained.
The high affinity of streptavidin for biotin, with a K
a
of approximately 2.5×10
13
M
−1
, has been advantageously utilized in many existing diagnostic and separation technologies, and in targeted drug/imaging agent delivery systems. However, the extremely high affinity of streptavidin for biotin can be detrimental in applications where reversible immobilization of streptavidin or biotinylated targets is ultimately desirable. An important example is affinity separations, where a biotinylated target molecule is captured with streptavidin and where subsequent release and recycling of the biotinylated target or capture agent (e.g. antibody) is desired. Similarly, in drug delivery applications where the streptavidin-biotin system forms the targeting and/or delivery component, the exceptionally slow biotin dissociation kinetics limits potential applications utilizing diffusion of the biotinylated imaging agent or drug to the therapeutic target, and may also result in slow in vivo clearance of biotinylated imaging agents.
In common with many other high-affinity protein-ligand systems, streptavidin utilizes three key molecular recognition mechanisms in its interaction with biotin: an extensive hydrogen bonding network, several direct aromatic side-chain contacts, and a flexible loop near the biotin binding site. Flexible loops are protein structural elements often found near the binding sites or active sites of receptors and enzymes. With many flexible loops, ligand binding is accompanied by a open-to-closed (or disorder-to-order) conformation change in going from the unbound to the ligand-bound state (Noble M E M et al. (1993) Proteins 16:311-326; Wierenga R K, et al. (1991) Proteins 10:33-49; Morton A, et al. (1995) Biochemistry 34:8576-8588; Tanaka T, et al. (1992) Biochemistry 31:2259-2265; and Falzone C J, et al. (1994) Biochemistry 33:439-442). The loops presumably play an important role in gating ligand association and dissociation, but their energetic contributions to molecular recognition remain unclear. The free energy of binding is the result of balancing the entropic costs/benefits of ordering of loops and release of bound water with the enthalpic benefits of burying non-polar surface area and establishing bonding contacts. It is expected that protein-ligand interactions will lead to energetic signatures similar to those associated with protein folding. Murphy K P, et al. (1993) Proteins 15:113-120; Spolar R S, et al. (1994) Science 263:777-784.
A prominent feature accompanying biotin association is the conformational change of a flexible binding loop (Hendrickson W A, et al. (1989) Proc Natl Acad Sci USA 86:2190-2194; Weber P C, et al. (1989) Science 243:85-88). A crystallographic study of the flexible loop in core streptavidin has been reported (Freitag S, et al. (1997) Protein Sci 6:1157-1166). The loop (residues 45-52 of SEQ ID NO:1) is in a closed conformation in the presence of biotin and in an open conformation in apo-streptavidin. Residues 49 through 52 (SEQ ID NO:1) are found in a 3
10
helix and the open conformation is stabilized by a hydrogen bonding interaction between residues 45 and 52 (SEQ ID NO:1). In a tetragonal crystal form, these residues are disordered in the open conformation (Weber P C, et al. (1989) Science 243:85-88). Ser45 (SEQ ID NO:1) terminates the &bgr;-strain leading into the loop and the side-chain oxygen of this residue is hydrogen-bonded to one of the ureido-oxygen of biotin, and the backbone amide nitrogen of Asn49 (SEQ ID NO:1) is hydrogen-bonded to the biotin carboxylate. The rearrangement and/or deletion of this loop can lead to many changes, particularly changes in binding of substrate. For many enzymes, it may be useful to alter binding characteristics, such as, for example, increasing or decreasing binding affinity.
Circular permutation is a technique wherein the normal termini of a polypeptide are linked and new termini are created by breaking the backbone elsewhere. In many polypeptides, the normal termini are in close proximity and can be joined by a short amino acid sequence. The break in the polypeptide backbone can be at any point, preferably at a point where the natural function and folding of the polypeptide are not destroyed. Circular permutation creates new C- and N-termini, allowing creation of fusion proteins wherein the fused peptide or protein is attached at a different place on the host protein. For example, if the natural termini are at the interior of the base protein, it may be disruptive to attach a peptide or protein at the natural termini, By changing the attachment location to a place near the exterior of the host protein, stability of the host protein may be maintained. In some situations, disruption of a loop near the binding site may advantageously disrupt substrate binding.
It would be advantageous to provide streptavidin mutants having a lower binding affinity for biotin than wild type streptavidin. It would be advantageous to provide streptavidin fusion proteins having a lower binding affinity for biotin than fusion proteins including wild type streptavidin.
It would be advantageous to provide streptavidin fusion proteins wherein the second peptide or protein is attached at a more useful position.
BRIEF SUMMARY OF THE INVENTION
Circularly permuted proteins are described wherein the natural termini of the polypeptide are joined and the resulting circular protein is opened at another point to create new C- and N- termini. The resulting protein exhibits some altered characteristic such as reduced substrate binding, for example. Fusion proteins can be made from the circularly permuted protein by attaching the second polypeptide to these newly created termini. These fusion proteins will have altered properties from a fusion protein made by attaching the second polypeptide to the natural termini. For example, the second peptide or protein can be attached at a position where it is more accessible to its substrate or intended target. In the preferred embodiment, the base polypeptide is streptavidin. Circular permutation of streptavidin results in a circularly permuted biotin binding protein. In one embodiment, a flexible polypeptide loop important for the binding of biotin was opened by creation of the circularly permuted protein. The original termini (residues 13 and 139 of SEQ ID NO:1) were joined by a linker. The biotin association constant was reduced approximately six orders of magnitude below that of wild type streptavidin to 10
7
M
−1
. Fusion proteins of the circularly permuted streptavidin can be made with secondary peptides/proteins such as IgG binding protein A or single-chain antibodies.


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