WO2001096530A2 - Streptavidines dimeriques - Google Patents

Streptavidines dimeriques Download PDF

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WO2001096530A2
WO2001096530A2 PCT/US2001/041027 US0141027W WO0196530A2 WO 2001096530 A2 WO2001096530 A2 WO 2001096530A2 US 0141027 W US0141027 W US 0141027W WO 0196530 A2 WO0196530 A2 WO 0196530A2
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streptavidin
ala
biotin
gly
thr
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PCT/US2001/041027
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WO2001096530A8 (fr
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Charles R. Cantor
Filiz M. Aslan
Sandor Vajda
Takeshi Sano
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The Trustees Of Boston University
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Priority to AU2001273594A priority Critical patent/AU2001273594A1/en
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Publication of WO2001096530A8 publication Critical patent/WO2001096530A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)

Definitions

  • This invention relates to recombinant streptavidin proteins that bind biotin, to recombinant streptavidin proteins having an altered affinity for binding biotin and to methods utilizing recombinant streptavidin proteins for the detection and isolation of targets.
  • the invention also relates to nucleic acids encoding recombinant streptavidin proteins and to recombinant cells which contain and express proteins encoded by these nucleic acids.
  • avidin and streptavidin have almost the same high affinity for biotin, they are different in many other respects.
  • the two proteins have different molecular weights, electrophoretic mobilities and overall amino acid composition.
  • Avidin is a glycoprotein found in egg whites and the tissues of birds, reptiles and amphibia.
  • streptavidin avidin has almost the same high affinity for biotin and exists as a tetramer with a molecular weight of between about 67,000 to about 68,000 daltons.
  • Avidin also has a high isoelectric point of between about 10 to about 10.5 and contains carbohydrates which cause it to bind non-specifically to biological materials including cell nuclei, nucleic acids and lectins.
  • Biotin also known as vitamin H or cis-hexahydro-2-oxo-lH-thieno-(3,4)- imidazole-4-pentanoic acid, is an essential vitamin found in every living cell including bacteria and yeast. In mammals, the tissues having the highest amounts of biotin are the liver, kidney and pancreas. Biotin levels also tend to be raised in tumors and tumor cells. In addition to cells, biotin can be isolated from secretions such as milk which has a fairly high biotin content. Biotin has a molecular weight of about 244 daltons, much lower than its binding partners avidin and streptavidin.
  • Biotin is also an enzyme cofactor of pyruvate carboxylase, trans-carboxylase. acetyl-CoA- carboxylase and beta-methylcrotonyl-CoA carboxylase which together carboxylate a wide variety of substrates. Only the intact bicyclic ring of biotin is required for the strong binding to streptavidin. The carboxyl group of biotin' s pentanoic acid side chain has little to contribute to this interaction. Consequently, biotin derivatives, reactive to a variety of functional groups, can be prepared by modifying the pentanoic acid carboxyl group without significantly altering the target's physical characteristics or biological activity. This allows biotin to be conjugated to a number of target molecules.
  • Streptavidin is produced by the bacteria, Streptomyces avidini, and exists as a tetrameric protein having four identical subunits.
  • the full length streptavidin monomer is 159 amino acids in length, some 30 residues longer than avidin. It contains no carbohydrate and has an acidic isoelectric point of about 5.0 which accounts, in part, for the low non-specific binding level.
  • Each subunit of streptavidin is initially synthesized as a precursor of 18,000 daltons which forms a tetramer of about 75,000 daltons. Secretion and post-secretory processing results in mature subunits having an apparent size of 14,000 daltons.
  • Processing occurs at both the amino and carboxyl termini to produce a core protein of about 13,500 daltons, having about 125 to 127 amino acids.
  • This core streptavidin forms tetramers and binds to biotin as efficiently as natural streptavidin.
  • the amino acid sequence of the mature 160 amino acid protein is as follows:
  • a natural streptavidin tetramer is formed by interdigitating a pair of streptavidin dimers with their dyad axes coincident (W. A. Hendrickson et al., Proc. ⁇ atl. Acad. Sci. USA 86:2190-94,1989).
  • a tetramer is stabilized by numerous van der Waals forces, with subunits forming a symmetric dimer additionally connected by hydrogen bonds near the carboxyl terninus.
  • the force distribution within a tetramer indicates that there are two classes of subunit interfaces. One interface is between subunits in a stable symmetric dimer and the other is between two stable dimers.
  • the mature streptavidin tetramer binds one molecule of biotin per subunit and the complex, once formed, is unaffected by most extremes of Ph, organic solvents and denaturing conditions. Separation of streptavidin from biotin requires harsh conditions, such as 8 M guanidine, pH 1.5, or autoclaving at 121. degree. C. for 10 minutes.
  • streptavidin-biotin binding systems are numerous.
  • the exceptionally high affinity and stability of the complex ensures complete reaction.
  • Biotin's small size allows it to be conjugated to most molecules with no loss in molecular activity.
  • Multiplicity of biotinylation sites combined with the tetramei ⁇ c structure of streptavidin allows for amplification of the desired signal.
  • the system is extremely versatile, as demonstrated by the large number of functional targets, binders and probes.
  • the system is amenable to multiple labelling techniques, a wide variety of biotinylated agents and streptavidin-containing probes are commercially available.
  • Streptavidin-biotin complexes are used in a number of diagnostic and purification technologies.
  • a target molecule to be purified or detected or otherwise targeted is bound either directly to biotin or to a biotinylated intermediate.
  • the binder may be almost any molecule or macromolecule that will complex with or conjugate to a target molecule. For example, if a particular antigen is the target, its binder would be an antibody.
  • the biotinylated target is bound to streptavidin which may be bound to a probe for ease of detection.
  • This basic technique is utilized in chromatography, cytochemistry, histochemistry, pathological probing, immunoassays, bioaffmity sensors and cross-linking agents, as well as more specific techniques such as targeting, drug delivery, flow cytometry and cytological probing.
  • the size of the streptavidin tetramer is still typically larger than ideal.
  • the large size can have disadvantageous pharmokinetics such as slow clearance from circulation and undesirable, nonspecific accumulation in organs like the kidney and liver.
  • streptavidin-biotin complexes The origins of the unusually high binding affinity seen in streptavidin-biotin complexes has not been fully elucidated. X-ray crystallographic studies have shown that streptavidin's carboxyl and amino termini lie on the molecule's surface (P. C. Weber et al, J. Am. Chem. Soc. 114:3197-200, 1992). These termini have been modified by cleavage or conjugation with a minimal effect on biotin binding affinity.
  • the streptavidin-biotin complex does not involve any covalent bonds, but does contain many hydrogen bonds, hydrophobic interactions and van der Waal interactions. These interactions are largely mediated by the aromatic side chains of tryptophan. Two tryptophan-lysine pairs are conserved between streptavidin and avidin. These pairs are found at positions 79-80 and 120-121 in streptavidin.
  • Trp-120 of one particular streptavidin subunit makes contact with the biotin bound to an adjacent subunit (A. Pahler et al.,J. Biol. Chem. 262:13,933-37,1987).
  • Lysine is known to play a critical role in avidin- biotin complex formation. For example, when an avidin lysine at positions 45, 94 or 111 is bound to a dinitrophenyl group, activity is abolished (Avidin-Biotin Chemistry: A Handbook, M. D. Savage et al., editors, page 7, 1992).
  • Trp-120 may play a role in maintaining local structures of streptavidin, particularly around the biotin-binding sites and the dimer—dimer interface. Strong hydrophobicity is observed around Trp-120 and three other tryptophan residues (Trp- 79, 92 and 108) that make contact with biotin (P. C. Weber et al, Sci. 243:85- 88,1989; C. E. Argara na et al, Nuc. Acids Res. 14:1871-82, 1986). In addition, hydrophobic interactions are the major force for the stable association of the two symmetric streptavidin dimers.
  • the streptavidin-biotin binding system is essentially irreversible.
  • the streptavidin- biotin bond is not affected by pH values between 2 to 13, nor by guanidine-HCl concentrations up to 8 M (neutral pH).
  • the half-life for spontaneous dissociation of the streptavidin-biotin bond is about 2.5 years.
  • the extremely strong binding of biotin to streptavidin means that biotinylated proteins can only be recovered from streptavidin supports under denaturing conditions. This sort of system is inappropriate for many procedures such as, one of its principal uses, the purification of delicate proteins.
  • Streptavidin-biotin cannot be used in sequential assays to detect specific types of biomolecules, macromolecular complexes, viruses or cells present in a single sample.
  • the high affinity necessitates the use of harsh chemical reagents, complex procedures, and careful monitoring of the reactions. This also limits both yields and the ability to fully automate such reactions.
  • a number of methods have been developed in an attempt to create a releasable streptavidin-biotin or avidin-biotin conjugate. These methods include partly monomeric avidin beads, N-hydroxysuccinimide-iminobiotin and biotin or streptavidin cleavage. Monomeric avidin beads are formed by denaturing tetrameric avidin and coupling the denatured protein to chromatography beads. Thus, the so-called monomeric avidin is really a mixture of monomeric, dimeric and tetrameric proteins that have a binding affinity distributed between the wild type affinity of 10.sup.15 M.sup.-l and the reduced affinity of 10.sup.8 M.sup.-l. Thus, monomeric avidin beads produce low yields because some of the biotinylated products are irreversibly bound. Furthermore, the density and capacity of monomeric avidin beads is low.
  • N-hydroxysuccinimide-iminobiotin is a guanido analog of NHS-biotin with a pH sensitive binding affinity for streptavidin.
  • the complete dissociation of NHS-iminobiotin from streptavidin occurs at low pH without the need for strong denaturants.
  • the drawback to the NHS-iminobiotin system is that binding requires a pH of 9.5 or greater, while release requires a pH of less than 4.
  • the use of NHS-iminobiotin is limited to those few molecules which are stable over a wide pH range.
  • Another method of release involves biotin cleavage of the binding partners, for example, of a cleavable biotin such as immunopure NHS-SS-biotin which is commercially available (Pierce Chemical Co.; Rockford, 111.).
  • NHS-SS-biotin consists of a biotin molecule linked through a disulfide bond and an N-hydroxysuccinimide ester group that reacts selectively with primary amines. Using this group, NHS-SS- biotin is linked to a target molecule and the biotin portion removed by thiol cleavage.
  • This complex approach is slow and of limited use since thiols normally disrupt native protein disulfide bonds.
  • cleavage leaves a reactive sulfhydryl group that tends to react with other components of the mixture.
  • thiol-containing nucleic acids will no longer hybridize, severely limiting their usefulness.
  • This streptavidin molecule is a dimer of streptavidin subunits, wherein each subunit contains a mutation of His 127 to prevent tetramer formation as well as mutations at the exposed subunit-subunit interface to reduce hydrophobicity.
  • Preferred sites that are hydrophobic and should be mutated to remove hydrophobicity include Trp 120 , Leu 124 , Val 125 in addition to His 127 .
  • This hydrophobicity can be reduced by substituting these amino acid residues with more hydrophilic amino acids with care being taken that the side group substituted does not affect steric considerations because one wants the molecule to be able to fold in a three-dimensional structure approximating the core biotin binding configuration of the wild-type molecule.
  • acidic amino acids such as asp, glu, uncharged amino acids such as ser, asn, gin, gly, etc.
  • Preferred substitutions are selected from the groups consisting of asp, glu, gly, ser and asn. More preferably asp, asn and ser.
  • Trp is substituted with Asp
  • Leu 124 is substituted with Asp
  • Asn Nal 125 is substituted with
  • the internal amino acids 1 through 15 can be deleted and the determinal amino acids from amino acid 130, preferably 133, still more preferably 139 can be deleted.
  • the streptavidin subunits can correspond to amino acids 1-159 with the above-mentioned substitution.
  • the subunit can correspond to amino acids 13 to 139, 13 to 138, 13 to 133, 16 to 133, etc.
  • the modified streptavidin molecule has an ability to bind biotin that is at least 50% and more preferably at least 60% and still more preferably at least 75% that of the wild-type streptavidin.
  • the subunits comprise a single chain molecule.
  • These dimeric streptavidin molecules can be used for any purpose the wild- type streptavidin can be used for.
  • it can be used in two-step immunotargeting.
  • the target is tagged with biotinylated antibody or other molecule containing a moiety that streptavidin will bind to.
  • the streptavidin which is conjugated to a second molecule such as an imaging agent or radiotherapeutic agent is used to bind to, for example, the biotinylated antibody to tag the target.
  • a second molecule such as an imaging agent or radiotherapeutic agent
  • the invention overcomes the problems and disadvantages associated with current strategies and designs and provides streptavidin proteins and peptides with increased stability and reduced or enhanced affinity for biotin, and methods for utilizing these streptavidin in detection, identification, isolation and purification techniques.
  • the molecules comprise amino acid sequence of streptavidin corresponding to positions 13 to 139, 14 to 138 or 16 to 133 of the wild type molecule.
  • nucleic acids which encode streptavidin proteins of the invention.
  • Recombinant nucleic acids may be encoded within plasmids and replicated, chemically synthesized, or transformed into eukaryotic cells.
  • Another embodiment of the invention is directed to cells which comprise nucleic acids that encode streptavidin proteins of the invention.
  • Cells may be prokaryotic or eukaryotic and may constitutively or inducible express recombinant streptavidin protein.
  • Another embodiment of the invention is directed to methods for detecting or purifying a target from a heterogenous mixture which contains the target.
  • the target to be purified is biotinylated with biotin or derivative of biotin and contacted to a support to which is attached to a streptavidin molecule of the invention.
  • the components of the heterogenous mixture can be removed and the target isolated.
  • target may be coupled with streptavidin and the support coupled with biotin.
  • the target can be purified after contact with the support.
  • Another embodiment of the invention is directed to methods for targeting a pharmaceutical agent to a cell wherein the agent is coupled to a streptavidin of the invention and the cell contains biotin. Such methods can be used to treat or prevent disorders such as infections and neoplasms.
  • kits which contain a streptavidin molecule of the invention and, optionally, additional reagents for the detection or isolation of target substances.
  • FIG. 1 is a schematic of expression vector for a streptavidin molecule containing amino acid substitutions to stabilize dimer formation.
  • FIG. 2 is a comparison of a variety of streptavidin molecules.
  • FIG. 3 shows local structures around the biotin-binding site of natural streptavidin.
  • FIG. 4 is a schematic illustration of the structures of four streptavidin constructs.
  • the present invention is directed to a streptavidin molecule containing two streptavidin subunits, to nucleic acid sequences which encode these streptavidin subunits and molecules, to recombinant cells which contain these sequences, to methods for detecting and isolating small molecules, macromolecules and cells with streptavidin and to kits which contain streptavidin molecules and subunits.
  • This streptavidin molecule is a dimer of streptavidin subunits, wherein each subunit contains a mutation of His 127 to prevent tetramer formation as well as mutations at the exposed subunit-subunit interface to reduce hydrophobicity.
  • Preferred sites that are hydrophobic and should be mutated to remove hydrophobicity include Trp 120 , Leu 124 , Val 125 as well as His 127 .
  • This hydrophobicity can be reduced by substituting the hydrophobic amino acid residues with more hydrophilic amino acids with care being taken that the side group substituted does not affect steric considerations because the resultant modified molecule should have a conformation approximating the wild-type structure.
  • acidic amino acids such as asp, glu, uncharged amino acids such as ser, asn, gin, gly, etc.
  • Preferred substitutions are selected from the groups consisting of asp, glu, gly, ser and asn. More preferably asp, asn and ser.
  • Trp 120 is substituted with Asp
  • Leu 124 is substituted with Asp
  • Asn Val 125 is substituted with Asp or Ser
  • His 127 is substituted with Asp.
  • the internal amino acids 1 through 15 can be deleted and the determinal amino acids from amino acid 130, preferably 133, still more preferably 139 can be deleted.
  • the streptavidin subunits can correspond to amino acids 1-159 with the above-mentioned substitution.
  • the subunit can correspond to amino acids 13 to 139, 13 to 138, 13 to 133, 16 to 133, etc.
  • the use of these subunits results in stable reduced size dimers that destabilize the dimer/dimer interfaces and therefore produce a dimeric protein instead of the wild type tetramer.
  • the resultant molecule will have a reduced ability to bind biotin when changes are made at Tip 120 but it will still bind biotin.
  • the molecule has an ability to bind biotin that is at least 50% and more preferably at least 60% and still more preferably at least 75% that of the wild-type streptavidin.
  • the molecule is made as a single chain molecule to enhance binding to.
  • the target is tagged with biotinylated antibody or other molecule containing a moiety that streptavidin will bind to.
  • the streptavidin which is conjugated to a second molecule such as an imaging agent or radiotherapeutic agent is used to bind to, for example, the biotinylated antibody to tag the target.
  • a second molecule such as an imaging agent or radiotherapeutic agent
  • the streptavidin-biotin binding system is an established fixture in biology due, at least in part, to the ability of streptavidin to non-covalently interact with biotin. This association is highly specific and quite strong with a binding constant of greater than 1015 M-l. This tight binding can in some instances limit the usefulness of conventional streptavidin-biotin systems. Although molecules and cells can be isolated from complex mixtures, removal of one or the other of the binding partners is difficult. Dissociation is preferably accomplished under very harsh conditions such as 6-8 M guanidine-HCl, pH 1.5. Not surprisingly, such conditions also denature, and thereby inactivate or destroy most target biological substances.
  • streptavidin behaves better than antibodies as, for example, the radioisotope carrier for two step in vivo radioimmune imaging and therapy, its size is still much larger than ideal.
  • the present invention constructs streptavidin molecules, are stable, reduced-size streptavidin mutants that destabilize the dimer-dimer interfaces so as to produce a dimeric protein instead of the wild-type tetramer. This dimeric streptavidin should speed up the delivery of radioactivity to the tumor targets. To be active in biotin-binding in a patient, however, a dimer must remain soluble in serum, and the individual monomers must remain joined, and not dissociate. This is accomplished by the present invention.
  • the smaller size of the dimeric streptavidin allows this protein to better serve than the wild-type tetramer in a range of functions such as in an improved in vivo diagnostic or therapeutic agent by reducing the disadvantageous pharmacokinetics of natural tetrameric streptavidin, such as slow clearance from the circulation and undesirable, non-specific accumulation in organs like the kidney and liver.
  • the loop removal increased the solvation free energy (-G s ) to the target value of greater than -34 kcal/mol, it increased the electrostatic repulsion energy between the monomers, which caused the dimer to become unstable.
  • the present invention uses a different strategy in which loop deletion was avoided.
  • loop deletion we mutated, for example, LI 24 and N 125 to more hydrophilic amino acids to increase the solubility of the two-chain dimeric protein.
  • This design has let us obtain soluble two-chain dimeric streptavidins, which are folded into a functional dimeric form without using biotin.
  • Radioactive isotopes are a fundamental step in the treatment of cancer, and one way to do this is by attaching radioactive isotopes. Once they are attached to the tumor, its location in the body can be determined by detecting the emitted radiation.
  • the most widely used method known as radioimmunotargeting, attaches radioisotopes to cells via antibodies.
  • antibodies which are large Y- shaped proteins, can be produced such that they ideally bind to the surface of a certain cell type only and can be radiolabelled in vitro. Then these radiolabelled antibodies are injected into cancer patients. Their large size, however, is disadvantageous due to slow diffusion to tumor tissue from the bloodstream and retention in the kidney for long periods. During this time, the patient will be exposed to potentially harmful radiation levels.
  • the streptavidin tetramer has a molecular mass of about 60,000 Daltons, while M r for a typical antibody is about 150,000 Daltons.
  • Pretargeting is a novel method of radioimmunotargeting, which takes advantage of the strong noncovalent bond between biotin and streptavidin. This strategy has been developed to eliminate the disadvantages that are caused by using radiolabelled antibodies. Pretargeting involves coating the surface of tumor cells with antibodies that are covalently conjugated to biotin. The main difference from radioimmunotargeting is the absence of radioisotopes in the biotinylated antibody.
  • the slow-moving biotinylated antibody can attach to the surface of the tumor. Since the antibody is not radioactive, there is no harmful radiation exposure to the patient. After the excess biotinylated antibodies have cleared the patient's kidneys and bloodstream, the tumor will remain coated with biotinylated antibodies. This concludes the pretargeting step.
  • the targeting step in which radiolabelled streptavidin is injected into the patient. Radiolabelled streptavidin binds tightly to the biotin residues of the biotinylated antibody, which is attached to the tumor. Once the radiolabelled streptavidin binds the biotinylated antibodies, the tumor will be coated with radioisotopes, thereby achieving the diagnostic/therapeutic objective.
  • Dimeric streptavidin will not have as high binding as tetrameric streptavidin due to mutation of W120D. However, one can further increase the affinity of dimeric streptavidin by producing single-chain streptavidin which can be used with a phage display system to select the mutants having optimized binding. There are many other alterations that can be made in the subunits to effect other properties of the molecule. For example, many stable streptavidin proteins remain associated with biotin under conditions which would cause dissociation of biotin from wild-type streptavidin.
  • core streptavidin peptides form macromolecules of protein that have a higher affinity for biotin than wild-type streptavidin or even "natural core streptavidin" (containing the amino acid sequence of streptavidin from positions 13 to 139).
  • Natural core binds to about 0.94 to about 0.96 molecules of biotin per subunit of streptavidin and pH 7.4
  • pH 7.4 At 6 M guanidine hydrochloride, pH 7.4, full-length and natural core streptavidin show about a 20% reduction in biotin binding (about 0.768 molecules biotin per subunit).
  • pH 1.5 these same proteins show about a 15% reduction in biotin-binding affinity (about 0.826 molecules biotin per subunit).
  • Stv-13 a core streptavidin peptide (containing the amino acid sequence from 16 to 133) that forms tetrameric protein, shows no significant reduction in biotin-binding. This stability may be due to the absence of two charged residues from the core (Glu 14 and Lys 134) as well as two polar residues (Ser 136 and Ser 139). Furthermore, at 6 M guanidine hydrochloride, pH 1.5, natural core retains only about 20% of its normal biotin-binding capability whereas Stv-13 retains over about 80%.
  • stable streptavidin proteins bind to at least about 0.80 molecules of biotin per subunit at 6 M guanidine, pH 7.4, and more preferably at least about 0.9 molecules of biotin per subunit at 6 M guanidine, pH 7.4. It is also preferable that stable proteins bind to 0.8 molecules of biotin per subunit at 6 M guanidine, pH 7.4, and 0.7 molecules of biotin per subunit at 6 M guanidine, pH 7.4. This enhanced binding to biotin may be due to the lack of steric hinderance caused by the presence of the amino and/or carboxyl terminal sequences. Neither of these sequences appear to be necessary for binding, but in fact may have in some way interfered with biotin binding.
  • hybrid tetrameric streptavidin proteins containing four core streptavidin peptides, two with aspartic acid and two with lysine at position 127 retain biotin more strongly than natural biotin under harsh conditions.
  • both wild-type and natural core streptavidin loose biotin binding ability more quickly with increased temperatures.
  • These streptavidin protein retains greater than about 90% of bound biotin at 60.degree. C, preferably greater than about 80% of bound biotin at 60.degree. C, and more preferably greater than about 50% of bound biotin at SO.degree. C.
  • Streptavidin protein with increased affinity for biotin may have a binding affinity of greater than about 10.sup.12 M.sup.-l or about 10.sup.14 M.sup.-l, preferably greater than about 10.sup.15 M.sup.-l , and more preferably greater than about 10.sup.16 M.sup.-l .
  • the streptavidin molecule's attachment to biotin can be disrupted more easily than the wild-type streptavidin-biotin bond.
  • the streptavidin- biotin bond involving streptavidin proteins of the invention may be disrupted through the addition of fairly low concentration of biotin or biotin derivatives (biotin analogs) or modifications.
  • concentration of biotin which can be used to disrupt the streptavidin-biotin bond using streptavidin proteins of the invention is between about 0.1 mM to about 10 mM or, preferably, between about 0.3 mM to about 2 mM.
  • elution may be performed in a high pH (e.g.
  • the streptavidin molecule is bound to a solid support, for example, to facilitate detection and isolation procedures.
  • Typical solid supports include the surfaces of plastic, glass, ceramics, silicone or metal. These components may be found in detection kits, biological sample analysis devices and environmental sampling aids. Particularly useful types of such components include beads (magnetic beads; Dynal), tubes, chips, resins, gels, membranes (e.g. porous membranes), monolayers, plates, wells, films, sticks or combinations of the surfaces.
  • Solid supports also include hydrogels which may be made of a variety of polymers such as acrylamide and hydroxyapatite, or biomolecules such as dextran, cellulose or agarose. Binding of streptavidin to surfaces may be accomplished in several ways.
  • a solid support may be derivatized with a moiety which can form a covalent bond with streptavidin, avidin or biotin.
  • many commercially available surfaces may be used to couple streptavidin, avidin or biotin.
  • Example of such surfaces include agarose, cross linked agarose, acrylamide, agarose and acrylamide combinations, polyacrylic, cellulose, nitrocellulose membranes, nylon membranes, silicon and metal. These surfaces may be further modified to contain a carboxyl or other reactive group for crosslinking.
  • Reagents suitable for crosslinking to solid surfaces include cyanogen bromide, carbonyldiimidazole, glutaraldehyde, hydroxysuccinimide and tosyl chloride.
  • streptavidin which may be used to facilitate coupling to a solid support is composed of a core streptavidin containing a plurality of cysteines at the protein's amino or carboxyl terminus, and preferably the carboxyl terminus.
  • the cysteines facilitate binding to, for example, a thiolated support.
  • the streptavidin molecules of the invention are coupled to another molecule such as an imaging agent, a radiopharmaceutical, a biological agent such as an antibody, an antigen, a hormone, a cytokine, a cell or a pharmaceutical agent (for in vivo use).
  • an imaging agent e.g., an imaging agent, a radiopharmaceutical, a biological agent such as an antibody, an antigen, a hormone, a cytokine, a cell or a pharmaceutical agent (for in vivo use).
  • a biological agent such as an antibody, an antigen, a hormone, a cytokine, a cell or a pharmaceutical agent (for in viv
  • Cells may be eukaryotic such as mammalian cells, prokaryotic such as bacterial cells, insect cells, parasitic cells, fungal cells or yeast cells.
  • Coupling may be through electrostatic interaction or by covalent modification of one or both coupling partners. Covalent modifications are fairly stable when, for example, the coupled agent is subjected to the a biological environment such as occurs on administration to a host such as a mammal.
  • nucleic acids which encode a streptavidin subunit of the invention.
  • Such nucleic acids may further comprise transcription or translational control regions to regulate transcription, translation or secretion of the recombinant protein. Control sequences can also be introduced to provide inducible expression. This is very useful as streptavidin is somewhat harmful to most cells.
  • Recombinant nucleic acids may be introduced into bacterial cells, for example, by transformation, or into mammalian cells, for example, by transfection. Recombinant cells can be used to produce large quantities of recombinant molecule as needed or to provide a continuous source of recombinant streptavidin to a biological system.
  • Recombinant cells which can support the expression of streptavidin molecules or peptides (subunits) on the invention include eukaryotic cells such as mammalian and yeast cells and prokaryotic cells such as bacteria.
  • the target may be directly biotinylated and complexed with the reduced substrate affinity streptavidin.
  • a binder that complexes with the target may be the biotinylated component.
  • the detectable probe may be bound to the streptavidin and the system may involve more than one detectable probe.
  • Both the target and the support may be biotinylated, and the two are complexed together with the reduced substrate affinity streptavidin. Many permutations are made possible by the variety of targets, binders and probes.
  • Another embodiment of the invention is directed to a method for detecting or purifying a target from a heterogeneous mixture which contains target.
  • the target is biotinylated using biotin or a biotin derivative or modification appropriate for the target.
  • Targets may be nearly any substance such as biological or inorganic substances.
  • Biological substances include proteins and protein precursors, nucleic acids (DNA, RNA, PNA, aptomers) and nucleic acid precursors (nucleosides and nucleotides), carbohydrates, lipids such as lipid vesicles, cells, biological samples and pharmaceuticals.
  • Typical proteins which are detectable in conventional streptavidin/biotin systems, and useful herein, include cytokines, hormones, surface receptors, antigens, antibodies, enzymes, growth factors, recombinant proteins, toxins, and fragments and combinations thereof.
  • Subcellular components may also be purified by linking a ligand, with an affinity to the component, to a streptavidin of the invention.
  • Proteins which can be purified include cell adhesion molecules, antibody antigens, receptors ligands and antibodies.
  • Specific affmnity adsorbent moieties, such as wheat germ agglutinant, anti-idiotypic antibodies and dye ligands may be coupled to streptavidin to isolate glycosylated proteins such as SP1 transcription factor, dye binding proteins such as pyruvate kinase and liver alcohol dehydrogenase, and other antibodies.
  • glycosylated proteins such as SP1 transcription factor, dye binding proteins such as pyruvate kinase and liver alcohol dehydrogenase, and other antibodies.
  • cellular and subcellular organelles may be rapidly purified using specific antibodies.
  • the heterogenous mixture is contacted to the reduced-affinity streptavidin which may be fixed to a surface of a support of free in solution. Mixture is removed or the support removed from the mixture and the target purified. Alternatively, target may be coupled to streptavidin and biotin attached to the support. In either situation, the result is the same. However, using reduced-affinity streptavidin coupled to target, target may isolated free of any biotin.
  • a streptavidin with increased affinity for biotin may be used. This may be useful, for example, where the targeted substance is, for example, a malignant cell or a contaminant.
  • the contaminant may be removed by the increased affinity streptavidin and disposed of.
  • the increased affinity will ensure a more complete recovery than wildtype streptavidin. Because wildtype streptavidin is unstable under the extremes conditions of pH, salt, detergent, and disrupting agents, it is preferable to neutralize these agents before separation with streptavidin.
  • Increased affinity streptavidin may reduce or eliminate the need for neutralization. This reduction or elimination will reduce processing time and complexity and contribute directly to cost reduction.
  • targets can be transferred from one support to another using a manual or automated apparatus.
  • Sequential detection or purification techniques can also be used to purify targets to homogeneity. Such techniques were heretofore not possible when the streptavidin biotin bond could not be easily broken.
  • nearly any conventional detection or isolation methodology can be performed with conventional streptavidin-biotin procedures.
  • Another embodiment of the invention is directed to a method for the detection of a disorder in a patient such as a human.
  • Reduced-affinity streptavidin is naturally targeted to biotin. Biotinylation of a site within the body of the patient, such as, for example, using monoclonal or polyclonal antibodies coupled with biotin and specific for the site will target the coupled complex to that site.
  • Reduced-affinity streptavidin may be coupled with a pharmaceutical which can be used to treat the disorder.
  • Treatable disorders include neoplasms, genetic diseases and infections (e.g. viral, parasitic, bacterial, fungal).
  • Another embodiment of the invention is directed to a method for the isolation and culture of infectious agents from a patient.
  • Body fluids such as blood of a patient may be contacted with a support with antibodies specific for viral surface antigens. If the antibody was crosslinked to the solid support by a reduced-affinity streptavidin, bound infectious agents may be released without harm with a gentle elution technique.
  • the isolated agents may be definitively identified by live culture.
  • Infectious agents which can be isolated by this technique include slow viruses, malaria and infectious yeast.
  • nucleic acids can be immobilized to a column through a reduced affinity streptavidin complex.
  • the immobilized nucleic acid may be single or double stranded and it may comprised cloned sequence or random sequence.
  • the column may be used to enrich for nucleic acid-binding proteins.
  • the proteins bound to nucleic acids may be released without the use of nuclease or protease.
  • the product may be studied, without the disruption of the protein nucleic acid bond by native gel electrophoresis (a gel mobility shift assay).
  • Site directed mutagenesis without disturbing local environments around this residue can be accomplished by known means.
  • Trp-120 is mutated to a codon encoding Asp.
  • pTSA-13 which carries the coding sequence for amino acids 16-133 of mature streptavidin can be used as the starting material to make the modified streptavidin (FIG. 1).
  • a phosphoxylated oligonucleotide of the desired sequence is used to mutate the codon TGG encoding for Trp on residue 120 to a condon encoding Asp, e.g., GAC.
  • the reaction is initiated by hybridizing 10 pmoles of the phosphorylated oligonucleotide to the single stranded streptavidin DNA in a 10 .mu.l reaction with 20 mM Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 50 mM NaCl and 1 mM dithiothreitol (DTT).
  • Elongation and mutation is initiated by the addition of 10 .mu.l of 20 mM Tris-HCl, pH 7.5,10 mM MgCl.sub.2, 10 mM DTT, 2 mM dATP, 2 mM dTTP, 2 mM dCTP, 2 mM dGTP, 10 mM ATP, 5 units bacteriophage T4 DNA ligase and 2.5 units of Klenow.
  • This procedure is performed according to the in vitro mutagenesis kit supplied by Amersham. Subsequent procedures followed as recommended by Amersham. Resulting products created are used to transform competent E. coli cells. To select clones contained the desired mutations, the sequence was confirmed using a dideoxy chain termination procedure. The same technique is used to alter the other sites.
  • the mutated streptavidin of Example 1 is used to produce large quantities modified streptavidin protein. Because the expression of streptavidin in bacteria has a lethal effect to a cell, an inducible system is used.
  • the DNA fragment comprising the sequence encoding the streptavidin mutant is excised from its vector with the restriction endonucleases Nde I and BamH I, and cloned into the same sites in the T7 expression vector pET-3a. Resultant plasmids are transformed in BL21(DE3) (pLysE) bacteria.
  • BL21(DE3)(pLysE) cells carrying the expression plasmid are grown at 37. degree. C. in LB supplemented with 0.4% glucose, 150 .mu.g/ml ampicillin and 25 .mu.g/ml chloramphenicol until cultures reach a density of 0.6 at A.sub.600.
  • Expression of the Phe-120 streptavidin is induced by the addition of a gratuitous inducer, IPTG, to a final concentration of 0.4 mM.
  • Modified streptavidin is expressed for five hours at 37.degree. C. before the cells are harvested.
  • Modified streptavidin protein produced by induced E. coli is purified.
  • Cells expressing the mutant streptavidin are harvested by centrifugation at 1600.times.g for 10 minutes. Protein is purified from the insoluble fraction of cell extracts. Briefly, cells are pelleted, washed with an isotonic solution of 100 mM NaCl, 1 mM EDTA and 10 M Tris, pH 8.0, and resuspended in a detergent solution of 2 mM EDTA, 30 mM Tris-HCl, pH 8.0, 0.1% Triton X-100. Lysis occurrs under these conditions because the presence of T7 lysozyme in the cells.
  • Nucleic acid in the extract is digested for 15 minutes at room temperature by the addition of MgSO.sub.4, DNase I and RNase A, to final concentrations of 12 mM, 10 .mu.g/ml and 10 .mu.g/ml, respectively.
  • the insoluble fraction of the extract containing Phe-120 streptavidin is isolated by centrifugation of the nuclease treated extract at 39,000.times.g for 15 minutes. Pellets are washed with 2 mM EDTA, 30 mM Tris-HCl, pH 8.0, and 0.1% Triton X-100, and solubilized in 6 M guanidine hydrochloride, pH 1.5.
  • Impurities are removed by dialysis against 6 M guanidine hydrochloride pH 1.5. Mutant streptavidin are renatured slowly by dialysis against 0.2 M ammonium acetate, pH 6. After renaturation, insoluble impurities are removed by centrifugation at 39,000.times.g. Supernatant containing the mutant streptavidin protein are removed and collected. Final purifications are performed by 2-iminobiotin affinity chromatography. Polyacrylamide gel electrophoresis analysis (PAGE-SDS) of the modified streptavidin can be performed on protein expressed in E. coli carrying pTSA-38. Total cell protein of BL21(DE3)(pLysE), with or without pTSA-38, is analyzed using a 15% polyacrylamide gel.
  • PAGE-SDS Polyacrylamide gel electrophoresis analysis
  • Modified streptavidin can also be analyzed by SDS-PAGE. Briefly, approximately 3 .mu.g of modified streptavidin is applied to a 15% polyacrylamide gel. The right lane contains molecular mass standard proteins. The molecular weight of the protein estimated to be approximately 13,000 daltons, which is consistent with the molecular mass obtained from the deduced amino acid sequence (12,600 daltons).
  • biotin-binding affinities of wild type and modified streptavidin are determined by an equilibrium dialysis method using a micro dialyzer (Hoeffer Scientific).
  • D-[carbonyl-.sup.l4 C] biotin (2 nM-4 .mu.M; 53 mCi/mmol; Amersham)
  • 100 .mu.l of streptavidin (5.3 .mu.g/ml, 0.42 .mu.M subunits) are prepared separately in TBS (150 mM NaCl, 20 mM Tris-HCl, pH 7.4,0.02% NaN.sub.3) solutions.
  • the solubility of each core streptavidin molecule with and without biotin can be investigated by varying the concentration of ammonium sulfate or ethanol in the solution.
  • Biotin-binding sites of each streptavidin molecule are saturated by adding an equimolar amount of D-[carbonyl-.sup.l4 C]biotin, prior to the addition of an ammonium sulfate solution.
  • the amount of soluble streptavidin in the final supernatant is estimated from the radioactivity derived from bound biotin, determined by liquid scintillation counting.
  • the three mutants were designed by molecular modeling using effective binding free energy calculations. In vitro site-directed mutagenesis was used to construct three different genes for two-chain dimeric streptavidin.
  • Expression vectors encoding putative two-chain dimeric streptavidin were isolated and used to transform E. coli lysogens, BL21(DE3)(pLysS) and BL21(DE3)(pLysE) by known techniques. These strains carry the T7 RNA polymerase gene under the lacUN5 promoter in the chromosome, and these are used for high-level expression of genes cloned into expression vectors containing bacteriophage T7 promoter. Each dimeric streptavidin mutant was expressed efficiently in E.

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Abstract

L'invention concerne des protéines de streptavidine recombinantes se liant à la biotine, des protéines de streptavidine recombinantes présentant une affinité altérée pour se lier à la biotine, ainsi que des méthodes d'utilisation de protéines de streptavidine recombinantes en vue de la détection et de l'isolation de cibles. L'invention se rapporte également à des acides nucléiques codant pour ces protéines de streptavidine recombinantes, ainsi qu'à des cellules recombinantes contenant et exprimant les protéines codées par ces acides nucléiques.
PCT/US2001/041027 2000-06-16 2001-06-18 Streptavidines dimeriques WO2001096530A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008053892A1 (de) 2008-10-30 2010-05-06 Fachhochschule Gelsenkirchen Medizinisches Implantat mit biofunktionalisierter Oberfläche

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008053892A1 (de) 2008-10-30 2010-05-06 Fachhochschule Gelsenkirchen Medizinisches Implantat mit biofunktionalisierter Oberfläche

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