Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

• N-terminal sequences of proteins and peptides are most commonly determined using repeated cycles, either manual or automated, of the Edman degradation reaction. Each degradation cycle consists of three steps: coupling, cleavage and conversion. In the coupling step, the unmodified N-terminus of a peptide or protein is modified with phenylisothiocyanate under basic conditions to generate a phenylthiocarbomyl peptide.
• the PTC-N-terminal residue is cleaved from the polypeptide by either liquid or gaseous trifluoroacetic acid to form an anilinothiazoline-amino acid derivative of the original N-terminal residue and a protein or peptide whose penultimate residue now lies at the N-terminus.
• the ATZ-amino acid is unstable and is converted in the third step by acid to a more stable phenylthiohydantoin (PTH) -amino acid.
• PTH phenylthiohydantoin
• protein samples are normally adsorbed or coupled to a membrane or glass fiber support that lies in a reaction cartridge.
• the ATZ-amino acid is washed from the reaction cartridge into a conversion flask where it is converted into the PTH-amino acid.
• the PTH-amino acid is then injected into a HPLC column, and the amino acid is detected by UV absorbance and is identified by its retention time.
• the sensitivity of such analyzers can be a s low as 1 pmol of PTH-amino acid.
• the number of residues which can be sequentially identified is dependent upon the sample amount and on the sequence itself.
• the sample must be at least 80% pure. Mixtures of proteins generate multiple PTH-amino acids during each cycle of the Edman degradation. Thus, if multiple proteins are present in similar quantities in a sample, it is almost impossible to assign a sequence to a particular protein. In addition, if the N-terminus is blocked, then sequences can arise form contaminating proteins. Second, the sample must be free of contaminants such as Tris, glycine, sodium dodecyl sulfate (SDS) , or acrylamide which affect the performance of the instrument and create large artifactual peaks in the chromatograms. Finally, there must be sufficient quantity of sample available for analysis.
• SDS sodium dodecyl sulfate
• the epitope ordering procedure allows for characterization of post-translational modification events which occur on a protein.
• An object of the invention is to provide a method of identifying an amino acid sequence of a protein.
• This method comprises obtaining antibodies to amino acids.
• the antibodies are then modified by binding a first nucleotide sequence and a second nucleotide sequence to the antibody, the first nucleotide sequence comprising a modified 5' -nucleotide followed by a single restriction enzyme site and a long nucleotide sequence containing a base pair sequence at the 3' end capable of annealing to a complementary sequence, the second nucleotide sequence comprising the same modified 5'- nucleotide, single restriction enzyme site and long nucleotide sequence as the first nucleotide sequence but having the complementary base pair sequence at the 3' end.
• the modified antibodies are bound to a protein so that complementary base pair sequences at the 3' end of said first and second nucleotide sequences of the antibody form a primer template complex which is extended to form a stable double-stranded duplex.
• the double-stranded duplex comprising bound modified antibodies is treated with at least one restriction enzyme to digest the duplex and the digested products are separated. A band pattern produced from these digested products is detected and a portion of the amino acid sequence of the protein is identified.
• Another object of the present invention is to provide a method of characterizing post-translational events on a protein. This method comprises obtaining antibodies to at least two epitopes on a protein with at least one antibody being directed toward a post-translationally modified epitope.
• antibodies are then modified by binding a first nucleotide sequence and a second nucleotide sequence to said antibody, said first nucleotide sequence comprising a modified 5' -nucleotide followed by a single restriction enzyme site and a long nucleotide sequence containing a base pair sequence at the 3' end capable of annealing to a complementary sequence, said second nucleotide sequence comprising the same modified 5' -nucleotide, single restriction enzyme site and long nucleotide sequence as said first nucleotide sequence and having the complementary base pair sequence at the 3' end.
• the modified antibodies are then bound to a protein so that complementary base pair sequences at the 3' end of said first and second nucleotide sequences of said modified antibodies form a primer template complex which is extended to form a stable double-stranded duplex.
• the double-stranded duplex comprising bound modified antibodies is treated with at least one restriction enzyme to digest the duplex and the products of the digestion are separated. The band pattern produced from these digested products is detected and a post-translation event on the protein is identified and characterized.
• Another object of the present invention is to provide modified antibodies which comprise an antibody to a single, bi- or tri-amino acid or an epitope on a protein related to a post- translational event, a first nucleotide sequence having a modified 5' -nucleotide, a single restriction enzyme site, a long nucleotide sequence, and a first base pair sequence at the 3' end which is capable of annealing to a complementary sequence, and a second nucleotide sequence having the same modified 5' -nucleotide, single restriction enzyme site and long nucleotide sequence as the first nucleotide sequence and a second base pair sequence at the 3' end which is complementary to the first base pair sequence of the first nucleotide sequence, the first and second nucleotide sequences each being bound to the antibody.
• Figure 1 is an autoradiograph showing hybridization results for use of Abl4 and Abtaul-DNA-spanner complexes in detection of the tau protein.
• Lane 1 shows a mixture of the Abs which have not been mixed with tau but have been taken through the epitope ordering procedure.
• Lanes 2 and 3 show the results obtained from two different experiments by mixing tau protein with the Ab-DNA-spanner complexes and going through the epitope ordering procedure with no subsequent restriction enzyme digestion.
• the BamHI and EcoRI digestions of the material seen in lane 2 are shown in lanes 4 and 5, respectively.
• a technique has now been developed for the rapid and sensitive identification of amino acid sequences of unknown proteins. This technique can be used to facilitate the generation of nucleic acid hybridization probes. In addition, this technique can be used to characterize post-translational modification events such as amino acid phosphorylation, sulfation, and glycosylation occurring on proteins.
• antibodies are prepared against each of the 21 amino acids or to di-amino or tri-amino sequences.
• Antibodies can also be prepared against non-naturally occurring amino acids such as selenocysteine, homocysteine and homoserine.
• antibodies are prepared against epitopes on a protein relating to post-translational events. For example, antibodies have been prepared against the antigenic di-amino sequence phospho-tyrosine.
• monoclonal antibodies to the tau protein designated as antibody 14 (Abl4) and taul (Abtaul) are used.
• antibody is meant to refer to complete, intact antibodies, Fab fragments and F(ab) 2 fragments. It is preferred that each specific antibody is made into Fab fragments in accordance with methods well known in the art. The protein structure of complete intact antibodies, Fab fragment and F(ab) 2 fragments and the organization of the genetic sequences that encode such molecules are well known in the art.
• a first nucleotide sequence is prepared which contains a modified 5' -nucleotide followed by a single restriction enzyme site and a long nucleotide sequence which contains a base pair sequence at the 3' end which serves as a sequence to which a complementary sequence will anneal.
• a second nucleotide sequence is then prepared comprising the same modified 5' -nucleotide, restriction enzyme site and long nucleotide sequence as the first nucleotide sequence, but having a base sequence at the 3' end in the corresponding position which is complementary to the base pair sequence of the first nucleotide sequence.
• these DNA- spanning oligonucleotides comprises 198 base pairs with four distinct regions.
• the nucleotide sequence used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
• Equipment for such synthesis is sold by several vendors including Applied Biosystems (Foster City, CA) . Any other means for such synthesis may also be employed; the actual synthesis of the nucleotide sequences is well within the talents of the routineer.
• Oligonucleotides are synthesized using standard phosphoamidite chemistry and thus have a 5' -OH group. These oligonucleotides can be phosphorylated using a standard reaction and then derivatized to contain a 5'-NH 2 using the OligoLink Derivitzation Kit (Pierce Chemical) .
• the 5' nucleotide can be modified with the addition of a cystamine containing the free primary amine and is ready to be linked to the antibody.
• a nucleotide spacer region is followed by a nucleotide restriction enzyme recognition sequence, which is followed by a second nucleotide spacer region, followed at the 3'-end by a specific nucleotide hybridization sequence.
• a 36 nucleotide spacer region is followed by a 6 nucleotide restriction enzyme recognition sequence, followed by a 141 nucleotide spacer region, followed by a specific 15 nucleotide hybridization sequence.
• the BamHl and EcoRl restriction sites have been utilized for the DNA-spanners designated BAM36- 198, BAM36-198op, RI36-198, and RI38-198op.
• the BAM and RI spanners have a specific 15 nucleotide sequence at the 3' end which will complement the 15 nucleotide sequence at the 3' end of the 198op sequences.
• the nucleotide sequences are attached to a selected antibody covalently using a primary amine cross-linking agent.
• the derivatized DNA-spanners were attached to the tau antibodies using glutaraldehyde in accordance with well known methods.
• other bifunctional crosslinking agents such as bis(sulfosuccinimidyl) suberate and dimethyl suberimindate are also useful for forming amide bonds with the primary amine on the 5' end of the nucleotide sequences and the e-amino group of a lysine residue on the antibody.
• Such agents are commercially available through companies such as Pierce
• Abl4 was complexed with BAM36-198 and BAM36-198op, while Abtaul was complexed with RI36-198 and RI36-198op using glutaraldehyde.
• the antibody-oligo conjugates are subsequently separated from the unincorporated oligonucleotides by protein A column chromatography, in accordance with well known methods.
• the protein containing fractions are detected by 280 nm absorbance readings and stored at 4°C.
• the modified antibodies are then added to a sample containing a protein of unknown sequence under conditions which discourage annealing of nucleotide sequences. By these conditions it is meant to include, but is not limited to high salt concentrations or mechanical stabilization of the nucleotide sequence interaction.
• the modified antibodies then interact with their specific epitopes to bind with them.
• the ability of Abl4 and Abtaul-DNA-spanner complexes to associate with purified Abtaul protein has been demonstrated for at least one set of conditions.
• T4 DNA polymerase, Klenow and dNTPs are added so that the complementary base sequences of the first and second nucleotide sequences, which are brought into proximity because of the position of the nearest bound modified antibody, form a primer template complex which is then extended to form a stable double-stranded duplex.
• Restriction mapping is used frequently to map and characterize DNA fragments by those of skill in the art. Restriction mapping is accomplished with individual enzymes in accordance with the restriction enzyme suppliers' recommended conditions. For example, EcoRI digestion of the Ab-DNA mixtures is performed by addition of the following reagents in their final concentrations to the mixture: 100 mM Tris pH 7.5, 7 mM MgCl 2 and 50 mM NaCl. The enzymatic digestion then proceed for two hours at 37°C. Restriction enzymes can be obtained from a variety of different vendors including Promega (Madison, WI) , New England Biolabs
• the cleaved fragments are then separated on the basis of their length differences by electrophoresis in agarose or polyacrylamide gels.
• Gels containing various percentages of polyacrylamide or agarose (0.5 to 4%) can be used in the separation.
• the gel is run under conditions which achieve the best separation of fragments.
• the negatively charged nucleotide sequences migrate toward the anode. This migration is retarded by a matrix which contains large pores that sieve nucleotide sequences in a fashion so that longer nucleotide sequences migrate more slowly than shorter nucleotide sequences.
• the sieving properties are proportional to the concentration of agarose or polyacrylamide in the gel with migration being more retarded at higher concentrations.
• the sieving is even further controlled by the extent of cross-linking in the polymer.
• the pores are very large and the primary resolution variable is the concentration of the agarose used.
• Buffers used in these methods are selected in accordance with their buffering capacity which is needed during electrolysis of the water that occurs during electrophoresis.
• undigested, EcoRI or BamHI-digested reactions are mixed with gel loading dye, heated, and electrophoresed on a 4% polyacrylamide gel until the bromphenol blue dye is at the bottom of the gel.
• the Ab-oligo-oligo-Ab complexes are transferred to a nitrocellulose membrane by electroblotting.
• the nitrocellulose membrane is dried and then prehybridized with a solution containing BSA and salmon sperm DNA to prevent non-specific binding of the probe to the membrane.
• the blot is then probed with a radioactively labeled oligonucleotide containing the sequence (TGG) 10 which will hybridize with the DNA in the spacer region of the DNA-spanner oligonucleotides.
• This probe is labeled at the 5' end using 5' -polynucle ⁇ tide kinase and 32 P ATP.
• FIG. 1 shows hybridization results for use of Abl4 and Abtaul-DNA-spanner complexes in detection of the tau protein. These Ab-DNA complexes can be hybridized because of free (unreacted) single stranded DNA-spanners on each antibody. Lane 1 shows a mixture of the Abs which have not been mixed with tau but have been taken through the epitope ordering procedure. Lanes 2 and 3 show the results obtained from two different experiments by mixing tau protein with the Ab-DNA-spanner complexes and going through the epitope ordering procedure with no subsequent restriction enzyme digestion.
• the BamHI and EcoRI digestions of the material seen in lane 2 are shown in lanes 4 and 5 respectively.
• the slower mobility of the Ab-DNA complexes seen in lanes 2 and 3 show that the epitope ordering procedure produced a higher molecular weight complex while the restriction enzyme digestions of this complex shown in lanes 4 and 5 show that the restriction enzyme sites which were predicted to be generated were indeed generated.
• the antibody can be detected by protein A or a second, labeled antibody in accordance with well known methods.
• the banding pattern generated by using a single or multiple restriction enzymes determines the identity of the antibody associated with that restriction site. This information is then used to determine the order of the antibodies and the corresponding sequence of the bound protein.
• Such techniques are well-known to those of skill in the art as evidenced by wide use of the Edman degradation where identification of only a few amino acids is required to be able to generate a probe for isolation of the corresponding clone.
• antibodies are prepared against at least two epitopes on a protein with at least one antibody being directed toward a post-translationally modified epitope. These antibodies are modified and contacted with a sample as previously described in the present application.
• the function of proteins are often dependent upon the existence of groups added after primary translation and folding. For example, phosphorylation of tyrosine residues often activate or deactivate a protein kinase or lead to protein-protein dissociation of one of the G-protein subunits in a G-protein coupled receptor.
• proteins modification which affect protein function, stability, cellular trafficking or targeting for degradation include, but are not limited to, glycosylation, ADP-ribosylation, myristoylation, palmitoylation and ubiquitination.
• antibodies can be obtained to epitopes resulting from post-translational modifications of the protein allowing for identification and characterization of post-translational modifications on a protein.
• Current protein sequencing techniques and oligonucleotides sequencing methodologies do not establish these side chain modifications. In fact, in Edman Degradation, such modifications can make sequencing of a protein very difficult, if not impossible.
• a crude cellular homogenate can be used in the present invention since the antibodies are specific to epitopes within a specific protein and the chances of two or more antibodies nonspecifically binding to the same protein are very small.
• Antibodies generated against single, bi- or tri-amino acids are generated by first coupling the antigen to a carrier protein.
• carrier proteins include, but are not limited to keyhole limpet hemocyanin and bovine serum albumin. This coupling is accomplished using any number of methodologies well known in the art including, but not limited to, glutaraldehyde, m-Maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide or bis-diazotized benzidine.
• the antigen-carrier complex is used as an immunogen to immunize rabbits in accordance with procedures well known in the art. Once appropriate antibody titers are reached, antibodies are isolated from the rabbit blood in accordance with published procedures.
• monoclonal antibodies can be generated by immunizing mice with the antigen-carrier complex and generating hybridoma cells lines.
• the tau monoclonal antibodies used in one embodiment of the invention have been described by Kosik et al. (1988) .
• Abl4 and Abtaul were derived from a mouse immunization with bovine tau protein. This immunogen was prepared by boiling a thrice-cycled microtubule preparation. The supernatant was fractionated on a Sepharose 4B column to obtain a preparation enriched in tau.
• antibodies to phospho-tyrosine are produced. Phosphorylated tyrosine is conjugated to the carrier hemocyanin and antibodies are produced in rabbits.
• Animals are injected with the antigen-carrier complex to produce a humoral response and an appropriate screening procedure developed.
• the sera from test bleed are used to develop and validate the screening procedure.
• production of the hybridoma may begin.
• animals are boosted with a sample of antigen.
• antibody-secreting cells are isolated from lymphoid tissue of the immunized animal and mixed with myeloma cells. The mixture is centrifuged to generate good cell-to-cell contacts and fused with polyethylene glycol (PEG) . Following the fusion, the cells are removed from the PEG, diluted in selective medium and plated into ultiwell tissue culture dishes.
• PEG polyethylene glycol
• Samples of the tissue culture supernatants are tested for the presence of selected antibodies about one week after the fusion. Hybridomas testing positive are grown and then single-cell cloned. Using this approach allows for the establishment of a continuous cell line as the source of a single type of antibody.
• Fab fragments are prepared from the antibodies in accordance with published procedures using either papain or pepsin digestion of the Ab molecule.
• the antibody In papain digestion, the antibody is cleaved at the N-terminal side of the disulfide bond which holds the heavy chains together.
• a solution of the IgG at 5 mg/ml in 100 mM NaAcetate (pH 5.5) containing cysteine (final concentration 50 mM) and EDTA (final concentration 1 mM) is prepared and aliquoted into test tubes.
• Ten micrograms of papain is added for each milligram of antibody in the tube. The tube is then incubated at 37°C for 10 hours. Following incubation, iodoacetamide is added to a final concentration of 75 mM and the solution is incubated for 30 minutes.
• pepsin digestion the antibody is cleaved on the
• CAA 12 -GGA-TCC- (CAA) 47 -GCT-GAT-GCT-GAT-GCT-3'
• CAA 12 -GAA-TTC- (CAA) 47 -GCT-GAT-GCT-GAT-GCT-3'
• CAA 5' - (CAA) 12 -GAA-TTC- (CAA) 47 -GCT-GAT-GCT-GAT-GCT-3'
• Oligonucleotides were phosphorylated in a reaction mixture containing 25 microliters oligo (200 ng/microliter) , 5 microliter 10X PNC buffer (500 mM Tris pH 8.0, 10 mM MgCl 2 ) , 5 microliter ATP (10 mM stock) , 10 microliters H 2 0, and 5 microliter polynucleotide kinase. The mixture was incubated for 30 minutes at 37°C. Phenol/chloroform extraction was then performed followed by ethanol precipitation in accordance with well known methods. Derivatization of these oligonucleotides is accomplished through use of the OligoLink derivatzation kit provided by Pierce Chemical.
• the protocol provided by Pierce uses approximately 5 micrograms oligo mixed with a derivatization reagent (cystamine) .
• the derivatized oligo is then bound to a matrix and reduced with DTT to generate a free disulfide bond in the center of the cystamine leaving a free sulfhydryl group.
• the oligo in its final form is eluted from the matrix using 10 mM TE (Tris-EDTA) buffer.
• Example 4 Covalent Attachment of DNA-spanner to Antibody
• the derivatized oligonucleotides are attached to the antibody using glutaraldehyde. Approximately, 100 micrograms of antibody are mixed with 10 micrograms of the derivatized oligos in phosphate buffered saline (PBS) . A total volume of 200 microliters to 1 ml is appropriate. An equal volume of freshly made 0.1% EM grade glutaraldehyde (in PBS) is added and the mixture incubated at room temperature for 3 hours. 1/20 solution volume of 1 M ethanolamine pH 7 is added to the solution and incubated at room temperature for 2 hours.
• PBS phosphate buffered saline
• the antibody-oligo complexes are adjusted to pH 8.0 by addition of 1 M Tris pH 8 to a final concentration of 0.1 M.
• the solution is passed over protein A beads (BioRad Corp.) and the column washed with 5 volumes 0.1 M Tris pH 8, then 5 volumes 10 mM Tris pH 8.
• the antibody-oligo complex is eluted by adding 500 microliters 100 mM glycine pH 3 in 50 microliter aliquots. These aliquots are collected from the column into Eppendorf tubes containing 40 microliters 1 M Tris pH 8.
• the protein containing fractions are detected by absorbance readings at 280 nm.
• the protein containing fractions are pooled and stored until use at 4°C in 50% glycerol and 0.02% sodium azide.
• 10 microliters of 10X binding/reaction buffer (2 M Tris OAc pH 7.4, 1% gelatin, 2%
• 1 microliter 10X binding/reaction buffer 1.5 microliters 1 M MgOAc, 4 microliters of a mixture of 250 micromolar each dATP, dGTP, dCTP, and TTP, 1 microliter H 2 0, 3 microliters T4 DNA Polymerase (2 U/microliter) , 1 microliter Klenow (8 U/microliter) were added and the mixture incubated at room temperature for 30 minutes.
• the reactions were then digested with the appropriate restriction enzymes.
• the ion concentration was adjusted with the addition of 1.5 microliters 1 M KOAc and 3 microliters restriction enzyme (BamHI and EcoRI) .
• the tubes were then incubated at 37° for 30 minutes.
• the samples are mixed with gel loading dye, heated to 60°C for 5 minutes and electrophoresed on a 4% polyacrylamide gel (Laemmli, Nature 1970, 227: 680-685); 42-1 acrylamide: bis-acrylamide. After the bromphenol blue dye reaches the bottom of the gel, the complexes are transferred to nitrocellulose membrane by electroblotting.
• the nitrocellulose is baked at 80°C under vacuum for 4 hours.
• the blot is prehybridized for 4 hours at 40°C in 6X SSC (0.9 M sodium citrate, 0.09 M sodium chloride); 100 microgram/ml salmon sperm DNA, 5X Denhardt's (0.1% BSA, 0.1% polyvinylpyrrolidone, 0.1% ficoll) and 50% formamide.
• 6X SSC 0.9 M sodium citrate, 0.09 M sodium chloride
• 5X Denhardt's 0.1% BSA, 0.1% polyvinylpyrrolidone, 0.1% ficoll
• the blot is then hybridized at 50°C for 18 hours with 32 P-radiolabeled
• TGG 10 This probe is labeled using 5' -polynucleotide kinase where the reaction volume is 10 microliters and 3 microliters of 32 P-ATP (3000 Ci/mM, Amersham) is utilized in the reaction mix. After hybridization, the blot is washed twice for 30 minutes each with 2X SSC at 50°C followed by washing twice with 0.1X SSc at 50°C for 30 minutes each. The blot is exposed to X-ray film next to an intensifying screen for 24 hours.
• SEQ ID NO: 1 CAACAACAAC AACAACAACA ACAACAACAA CAACAAGGAT CCCAACAACA 50 ACAACAACAA CAACAACAAC AACAACAACAACAACAACAA CAACAACAAC 100 AACAACAACAACAACAA CAACAACAAC AACAACAACAACAACAACAA 150 CAACAACAAC AACAACAACAACAACAA CAAGCTGATG CTGATGCT 198 (2) INFORMATION FOR SEQ ID NO: 2:

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