MXPA00007931A - Biotin-binding receptor molecules - Google Patents

Abstract

A novel transmembrane protein is capable of binding to biotinylated molecules, the protein comprising a cytoplasmic domain, a membrane-spanning domain and an extracellular domain, wherein the extracellular domain comprises biotin-binding activity.

Description

RECEIVING MOLECULES LINKING BIOTIN DESCRIPTION OF THE INVENTION This invention describes proteins that encompass membranes having biotin-binding activity. Biotin (vitamin H) is a substance readily soluble in water that is found at low concentrations in the blood and tissues. The biological role of biotin is as an activated C02 carrier that allows the transfer of C02 to acceptors without the need for additional free energy. Activated carboxybiotin binds normally to an enzyme that is required for the formation of carboxybiotin. For example, biotin can bind to pyruvate carboxylase which, in the presence of acetyl CoA, catalyzes the formation of carboxybiotin and the subsequent transfer of the activated carboxyl group to pyruvate to form oxaloacetate. Biotin also binds with one of the highest naturally occurring affinities for avidin, a 63 kDa glycoprotein in chicken egg white, and streptavidin, a non-glycosylated protein of the bacterium Streptomyces avidinii. The bond is almost irreversible in character (Ka 1015 mol-1). The affinity between avidin and biotin has proven to be very useful in a wide variety of bioanalytical applications. For example, the avidin-biotin complex has been used successfully in a wide variety of detection systems where target molecules combine with biotin through its carboxy terminus to form biotinylated molecules that can be easily detected or separated from the solution . Biotinylation can occur without changing the biological or physiochemical properties of the various molecules and without affecting the binding capacity of the prosthetic group biotin to avidin. It has now been understood that the biotin binding activity of avidin and streptavidin can be used in the production of transmembrane proteins capable of binding biotinylated molecules. The proteins of the present invention may comprise a cytoplasmic domain, a membrane-spanning domain and an extracellular domain, wherein the extracellular domain comprises biotin-binding activity. The extracellular domain may comprise functional activity of avidin or streptavidin. By using proteins or nucleic acid molecules of this invention it is possible to signal the biotinylated molecules at specific sites of tissues. Molecules constructed in this way can be taken up by tissues or cells by endocytosis, allowing the molecules to exert their effects in or on the cell. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of a fusion protein of the present invention, wherein A represents avidin and B represents the membrane-spanning domain of an endocytotic receptor (and C represents biotin); Figure 2 is a schematic illustration of a cloning strategy using a binding vector; and Figure 3 is a schematic illustration of a cloning strategy using a retrovirus vector. The proteins of the present invention can be produced using conventional recombinant DNA technology. Typically, a DNA sequence encodes the functional domain of a biotin-binding protein such as avidin, streptavidin or a related protein, is designed within a genetic construct which comprises a DNA sequence encoding a protein having properties encompassing the membrane. Examples of avidin and streptavidin related proteins include AVR-1-AVR-5, AVR-X-AVR-V, Stv1 and Stv2. The individual domains of the fusion protein can be amplified by polymerase chain fusion or isolated from the parent cDNA using restriction enzymatic digestion, isolation and purification, for example using gel electrophoresis, and subsequent ligation, for example using DNA ligase. The fusion protein construct can then be transfected into any suitable host cell, cultured and isolated using standard protein purification techniques. The construct can also be used as unprotected DNA or as a complex plasmid / liposome, plasmid / polyethyleneimine, plasmid / dendrimer or plasmid / peptide. Alternatively, the construct can be introduced into a replication defective virus which can be used to signal that the construct specifies in vivo sites. For example, the construct can be a retroviral vector comprising the cDNA appropriate for the fusion protein. A replication-deficient retrovirus, e.g., Moloney murine retrovirus, can then be used for stable transfection of target cells and tissues. Other viruses that can be used include replication-deficient adenoviruses, adeno-, herpes virus, papilloma virus and synibis virus. The additional viruses will be apparent to those skilled in the art. In addition to the functional domains of avidin, streptavidin or related proteins, the fusion protein will typically comprise the domains encompassed by the membrane of the endocytotic receptors. The use of these receptors allows the capture of biotinylated molecules within a target cell. Suitable receptors that can be used in this invention include the class A scavenger receptor, low density lipoprotein receptor, very low density lipoprotein receptor, transferrin receptor and the LOX-1 receptor. The fusion protein may also comprise a linker between the receptor protein and the avidin peptide sequences. The binder can be of any length, with the proviso that the functional activity of the different components of the fusion protein is retained. In general, the fusion between the avidin or streptavidin peptide sequences and the peptide sequences of the receptor is between the extracellular domain of the receptor protein and any site outside the biotin binding site of avidin or streptavidin. The following example illustrates the invention. Example A DNA construct was created between the class A screener (ScR) of cattle (Kodama et al (1990) Nature 343: 531-535) and avidin (Green (1975) Adv. Prot. Chem. 2_9: 85- 133), which codes for a protein having a cytoplasmic domain ScR, domain spanning the membrane and an a-helical coiled domain, linked to a biotin binding domain. The complete amino acid sequence of the fusion protein is shown in SEC. FROM IDENT. No 2 where amino acids 1-53 represent the cytoplasmic domain; amino acids 55-79 represent the transmembrane DE domain; amino acids 81-111 represent a spacer domain; and amino acids 113-272 represent the a-helical coiled domain. Amino Acids 273-400 represent the mature avidin peptide sequence derived from the avidin cDNA (Gope et al (1987) Nucleic Acid Res. 1_5: 3595-3606) lacking a secretion signal. Briefly, the cDNA for ScR was obtained from cultured cells previously transfected with a plasmid (PLScRNL) containing the ScR cDNA with an internal Rous Sarcoma Virus promoter and Jin III restriction sites. The isolated cDNA was then inserted into a phyllIII site of the retroviral vector pLSlARNL. The avidin cDNA was produced by the polymerase chain reaction and then inserted into the retrovirus vector at the Sty 1 restriction site in the ScR cDNA. The cDNA containing the invention is shown as SEC. FROM IDENT. No. 1, wherein nucleotides 1-989 represent a long terminal repeat of Mo-MuSV; nucleotides 1071-2270 represent the region encoding the fusion protein; nucleotides 2376-3101 represent an untranslated region of the bovine cleansing receptor I cDNA; nucleotides 3107-3376 represent a promoter region of RSV; nucleotides 3727-4522 represent a neo gene. R; and nucleotides 4540-5177 represent a long terminal repeat of Mo-MuLV.

Figures 2 and 3 refer to the processes used in this Example. More specifically, Figure 2 shows how the ScR cDNA with an internal RSV promoter was cut from the plasmid pLScRNL by HindIII and cloned into a HindI II site of a binding vector. Figure 3 shows how the ScR-avidin-RSV cDNA was cloned into a HindI I I site of the retroviral vector pLRNL. Expression of the fusion protein in cells transfected with the vector can be confirmed by Northern staining and immunocytochemistry staining with an antibody raised against avidin. The experiments revealed that the complete mRNA transcriptor was translated into 55 kDa monomers, which was able to form secondary structures of 100 kDa dimers bound by S-S bonds under non-reducing conditions. Approximately 110 kDa dimeric and 55 kDa monomeric peptides were detected, using denaturing conditions. The result is comparable to the computer calculation for the monomeric fusion protein, 45 kDa. Under non-denaturing conditions (ie, using acetylation before Western staining), the strongest signal was approximately 220 kDa which was denatured to a dimer of approximately 110 kDa and a 55 kDa monomer, suggesting tetramer formation. The presence of the 220 kDa protein was also verified using chemical interlayers, for example 'NHS esters. The results show that avidin remains soluble and is capable of forming tetramers even when they are bound to the domains that span the membrane of the endocytotic receptors. The fusion protein was shown to be a functional protein capable of binding FITC-biotin when analyzed by confocal microscopy and atomic force microscopy. Non-transducing cells and cells transfected with a retrovirus vector containing the LacZ gene were used as controls. No non-specific binding of the biotin probes was detected to the LacZ transducer control cells by atomic force microscopy. As expected, the transfected cells showed specific binding that was repeatedly measurable in unfixed samples. The measured bond strengths were multiple of the average 149 + 19pN (mean + sd), which is, as was also expected, within the range of the 160 pN force of streptavidin-biotin binding reported previously (Florin et al (1994 ), Science 264: 415-417). The functionality of the construct can also be confirmed in vivo by showing the binding of the fluorescently labeled molecules to cells having the fusion protein construct, using FACS analysis. The functional activity of the fusion protein in vivo was analyzed in a rat model with malignant glioma. Cells with BT4C wild type glioma were implanted intracranially in the right corpus callosum at a depth of 2.5 mm in the brain of congenital BDIX female rats. Tumor growth is frequently monitored with high resolution MRI (magnetic resonance imaging). Three weeks after the inoculations of the tumor cells, retrovirus pseudotypes carrying cDNA for the fusion protein or LacZ gene at concentrations of 2 x 106 cfu / ml and 1.3 x 10 6 cfu / ml, respectively, were transferred into the tumor. first to a depth of 2.5 mm and then to a depth of 1.5 mm, with an interval of 10 minutes. The gene transfer was repeated after two days of culture. The animals were sacrificed and perfusion was fixed with 4% PFA 3 days after the last injection. The brains were removed and divided at the site of injection into two coronal pieces, sectioned on ice and analyzed with immunoreactivity against the anti-avidin antibody. The results show that the fusion protein was expressed in vivo in rat malignant glioma. The protein was detected in glioma cells and in ring-like structures that resemble the vascular cells of the endothelium in the blood vessels of the tumor.

LIST OF SEQUENCES 1) GENERAL INFORMATION: (i) APPLICANT: (ii) (A) NAME: Eurogene Limited (B) STREET: Marquis House, 67/68 Jermyn Street (C) CITY: London (E) COUNTRY: United Kingdom ( F) POSTAL CODE (ZIP): SW1Y 6NY (il) TITLE OF THE INVENTION RECEIVING MOLECULES THAT LINK IOTHINE (iii) NUMBER OF SEQUENCES: 2 (iv) METHOD OF READING ON THE COMPUTER: (A) TYPE OF MEDIUM: Disc soft (B) COMPUTER: compatible with an IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR THE IDENTITY SEQUENCE NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5177 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: Simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: DNAc (ix) 'FEATURE: (A) NAME / KEY: CDS (B) Location: 1071..2270 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTTGAAAGAC CCCACCCGTA GGTGGCAAGC TAGCTTAAGT AACGCCACTT TGCAAGGCAT 60 GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA AAGAAACAGC 120 TGAATACCAA ACAGGATATC TGTGGTAAGC GGTTCCTGCC CCGGCTCAGG GCCAAGAACA 180 GATGAGACAG CTGAGTGATG GGCCAAACAG GATATCTGTG GTAAGCAGTT CCTGCCCCGG 240 CTCGGGGCCA AGAACAGATG GTCCCCAGAT GCGGTCCAGC CCTCAGCAGT TTCTAGTGAA 300 TCATCAGATG TTTCCAGGGT GCCCCAAGGA CCTGAAAATG ACCCTGTACC TTATTTGAAC 360 TAACCAATCA GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC CGCTCTCCGA GCTCAATAAA 420 AGAGCCCACA ACCCCTCACT CGGCGCGCCA GTCTTCCGAT AGACTGCGTC GCCCGGGTAC 480 CCGTATTCCC AATAAAGCCT CTTGCTGTTT GCATCCGAAT CGTGGTCTCG CTGTTCCTTG 540 GGAGGGTCTC CTCTGAGTGA TTGACTACCC ACGACGGGGG TCTTTCATTT GGGGGCTCGT 600 CCGGGATTTG GAGACCCCTG CCCAGGGACC ACCGACCCAC CACCGGGAGG TAAGCTGGCC 660 AGCAACTTAT CTGTGTCTGT CCGATTGTCT AGTGTCTATG TTTGATGTTA TGCGCCTGCG 720 TCTGTACTAG TTAGCTAACT AGCTCTGTAT CTGGCGGACC CGTGGTGGAA CTGACGAGTT 780 CTGAACACCC GGCCGCAACC CTGGGAGACG TCCCAGGGAC TTTGGGGGCC GTTTTTGTGG 840 CCCGACCTGA GGAAGGGAGT CGATGTGGAA TCCGACCCCG TCAGGATATG TGGTTCTGGT 900 AGGAGACGAG AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CGGTTTGGAA 960 CCGAAGCCGC GCGTCTTGTC TGCTGCAGCC AAGCTTGGGC TGCAGGTCGA CTCTAGAGGA 1020 TCAATTCGGC ACGAGTAAAT CGGTGCTGCC GTCTTTAGGA CATATGAAGT ATG GCA 1076 Met Ala 1 CAG TGG GAT GAC TTT CCT GAT CAG CAA GAG GAC ACT GAC AGC TGT ACA 1124 Gln Trp Asp Asp Phe Pro Asp Gln Gln Glu Asp Thr Asp Ser Cys Thr 5 10 15 GAG TCT GTG AAG TTC GAT GCT CGC TCA GTG ACÁ GCT TTG CTT CCT CCC 1172 Glu Ser Val Lys Phe Asp Ala Arg Ser Val Thr Ala Leu Leu Pro Pro 20 25 30 CAT CCT AAA AAT GGC CCA ACT CTT CAA GAG AGG ATG AAG TCT TAT AAA 1220 Bis Pro Lys Asn Gly Pro Thr Leu Gln Glu Arg Met Lys Ser Tyr Lys 35 40 45 50 ACT GCA CTG ATC ACC CTT TAT CTC ATT GTG TTT GTA GTT CTC GTG CCC 1268 Thr Ala Leu He Thr Leu Tyr Leu He Val Phe Val Val Leu Val Pro 55 60 65 ATC ATT GGC ATA GTG GCA GCT CAG CTC CTG AAA TGG GAA ACG AAG AAT 1316 He He Gly He Val Wing Ala Gln Leu Leu Lys Trp Glu Thr Lys Asn 70 75 80 TGC ACG GTT GGC TCA GTT AAT GCA GAT ATA TCT CCA AGT CCG GAA GGC 1364 Cys Thr Val Gly Ser Val Asn Wing Asp He Ser Pro Pro Pro Glu Gly 85 90 95 AAA GGA AAT GÍ3C AGT GAA GAT GAA ATG AGA TTT CGA GAA GCT GTG ATG 1412 Lys Gly Asn Gly Ser Glu Asp Glu Met Arg Phe Arg Glu Ala Val Met 100 105 110 GAA CGC ATG AGC AAC ATG GAA AGC AGA ATC CAG TAT CTT TCA GAT AAT 1460 Glu Arg Met Ser Asn Met Glu Ser Arg He Gln Tyr Leu Ser Asp Asn 115 120 125 130 GAA GCC AAT CTC CTA GAT GCT AAG AAT TTC CAA AAT TTC AGC ATA ACÁ 1508 Glu Ala Asn Leu Leu Asp Ala Lys Asn Phe Gln Asn Phe Ser He Thr 135 140 145 ACT GAT CAA AGA TTT AAT GAT GTT CTT TTC CAG CTA AAT TCC TTA CTT 1556 Thr Asp Gln Arg Phe Asn Asp Val Leu Phe Gln Leu Asn Ser Leu Leu 150 155 160 TCC TCC ATC CAG GAA CAT GAG AAT ATC ATA GGG GAT ATC TCC AAG TCA 1604 Be Ser He Gln Glu His Glu Asn He He Gly Asp He Ser Lys Ser 165 170 175 TTA GTA GGT CTG AAC ACC ACTA GTA CTT GAT TTG CAG TTC AGT ATT GAA 1652 Leu Val Gly Leu Asn Thr Thr Val Leu Asp Leu Gln Phe Ser He Glu 180 185 190 ACA CTG AAT GGC AGA GTC CAA GAG AAT GCA TTT AAA CAA CAA GAG GAG 1700 Thr Leu Asn Gly Arg Val Gln Glu Asn Wing Phe Lys Gln Gln Glu Glu 195 200 205 210 TTA GAG GAG CGT ATA TAC AAT GCA TCA GCA GAA ATT AAG 1748 Met Arg Lye Leu Glu Glu Arg He Tyr Asn Wing Being Wing Glu He Lys 215 220 225 TCT CTA GAT GAA AAA CAA GTA TAT TTG GAA CAG GAA ATA AAA GGG GAA 1796 Ser Leu Asp Glu Lys Gln Val Tyr Leu Glu Gln Glu He Lys Gly Glu 230 235 240 ATG AAA CTG TTG AAT AAT ATC ACT AAT GAT CTG AGG CTG AAG GAT TGG 1844 Met Lys Leu Leu Asn Asn He Thr Asn Asp Leu Arg Leu Lys Asp Trp 245 250 255 GAA CAT TCT CAG ACA TTG AAA AAT ATC ACT TTA CTC CAA GGT GCC AGA 1892 Glu His Ser Gln Thr Leu Lys Asn He Thr Leu Leu Gln Gly Wing Arg 260 265 270 • AAG TGC TCG CTG ACT GGG AAA TGG ACC AAC GAT CTG GGC TCC AAC ATG 1940 Lys Cys Ser Leu Thr Gly Lys Trp Thr Asn Asp Leu Gly Ser Asn Met 275 280 285 290 ACC ATC GGG GCT GTG AAC AGC AGA GGT GAA TTC ACA GGC ACC TAC ATC 1988 Thr He Gly Wing Val Asn Ser Arg Gly Glu Phe Thr Gly Thr Tyr He 295 300 305 ACÁ GCC GTA ACÁ GCC ACÁ TCA AAT GAG ATC AAA GAG TCA CCA CTG CAT 2036 Thr Wing Val Thr Wing Thr Ser Asn Glu He Lys Glu Ser Pro Leu His 310 315 320 GGG ACA CAA AAC ACC ATC AAC AAG AGG ACC CAG CCC ACC TTT GGC TTC 2084 Gly Thr Gln Asn Thr He Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe 325 330 335 ACC GTC AAT TGG AAG TTT TCA GAG TCC ACC ACT GTC TTC ACG GGC CAG 2132 Thr Val Asn Trp Lys Phe Ser Glu Ser Thr Thr Val Phe Thr Gly Gln 340 345 350 TGC TTC ATA GAC AGG AAT GGG AAG GAG GTC CTG AAG ACC ATG TGG CTG 2180 Cys Phe He Asp Arg Asn Gly Lys Glu Val Leu Lys Thr Met Trp Leu 355 360 365 370 CTG CGG TCA AGT GTT AAT GAC ATT GGT GAT GAC TGG AAA GCT ACC AGG 2228 Leu Arg Ser Ser Val Asn Asp He Gly Asp Asp Trp Lys Wing Thr Arg 375 380 385 GTC GGC ATC AAC ATC TTC ACT CGC CTG CGC ACÁ CAG AAG GAG 2270 Val Gly He Asn He Phe Thr Arg Leu Arg Thr Gln Lys Glu 390 395 400 TGAGTGAGTG ACCAAGGTCC TCCTGGACTC CAGGTGAAAA AGGAGATAGA GGCCCTCCTG 2330 GACAAAATGG TATACCAGGC TTTCCAGGTC TAATAGGTAC TCCAGGTCTT AAAGGTGATC 2390 GGGGGGGATCT CTGGTTTACC TGGAGTTCGA GGATTCCCAG GACCAATGGG GAAGACCGGG 2450 6 AAGCCAGGAC TTAATGGACA AAAAGGCCAG AAGGGAGAAA AAGGGAGTGG AAGCATGCAA 2510 AGACAATCTA ATACAGTCCG ACTGGTGGGT GGCAGCGGCC CTCACGAAGG CAGAGTGGAG 2570 ATTTTTCACG AAGGCCAGTG GGGTACGGTG TGTGACGACC GCTGGGAACT GCGTGGAGGA 2630 CTGGTCGTCT GCAGGAGCTT GGGATACAAA GGTGTTCAAA GTGTGCATAA GCGAGCTTAT 2690 TTTGGAAAAG GTACGGGTCC AATATGGCTG AATGAAGTAT TTTGTTTCGG GAAAGAGTCA 2750 TCCATTGAAG AGTGCAGAAT TAGACAGTGG GGTGTGAGAG CCTGTTCGCA CGACGAAGAT 2810 GCTGGGGGTC ACTTTGCACC TACATAATGC ATCATATTTT CATTCACATT TTTTAAACTG 2870 TTATAAAGTG ATTTTTTTTCC TTTGCTTCAC TAAAATCAGC TTAATTAATA TTTAAGAAAC 2930 TAAGAATTTT ATCCACAGAA AAGGAATATT TAAAAATCAC TGGATAAACA TATAAAATAG_2990_CTTCATATTT GCTTCAAATA CCAGAACCAT TTCAACTTCT CTAGGTTTTT AAGTGGCTCG 3050 TGCCGAATTG ATCCCCTCAG GATATAGTAG TTTCGCTTTT GCATAGGGAG GGGGAAATGT 3110 AGTCTTATGC AATACTCTTG TAGTCTTGCA ACATGGTAAC GATGAGTTAG CAACATGCCT 3170 TACAAGGAGA GAAAAAGCAC CGTGCATGCC GATTGGTGGA AGTAAGGTGG TACGATCGTG 3230 CCTTATTAGG AAGGCAACAG ACGGGTCTGA CATGGATTGG ACGAACCACT GAATTCCGCA 3290 TTGCAGAGAT ATTGTATTTA AGTGCCTAGC TCGATACAGC AAACGCCATT TGACCATTCA 3350 CCACATTGGT GTGCACCTCC AAGCTTCACG CTGCCGCAAG CACTCAGGGC GCAAGGGCTG 3410 CTAAAGGAAG CGGAACACGT AGAAAGCCAG TCCGCAGAAA CGGTGCTGAC CCCGGATGAA 3470 TGTCAGCTAC TGGGCTATCT GGACAAGGGA AAACGCAAGC GCAAAGAGAA AGCAGGTAGC 3530 TTGCAGTGGG CTTACATGGC GATAGCTAGA CTGGGCGGTT TTATGGACAG CAAGCGAACC 3590 7 GGAATTGCCA GCTGGGGCGC CCTCTGGTAA GGTTGGGAAG CCCTGCAAAG TAAACTGGAT 3650 GGCTTTCTTG CCGCCAAGGA TCTGATGGCG CAGGGGATCA AGATCTGATC AAGAGACAGG 3710 ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG 3770 GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 3830 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG 3890 TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT 3950 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG 4010 CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT 4070 CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 4130 CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA 4190 GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA 4250 GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA 4310 TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC 4370 GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 4430 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC 4490 CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA TAAAATAAAA 4550 GATTTTATTT AGTCTCCAGA AAAAGGGGGG AATGAAAGAC CCCACCTGTA GGTTTGGCAA 4610 GCTAGCTTAA GTAACGCCAT TTTGCAAGGC ATGGAAAAAT ACATAACTGA GAATAGAGAA 4670 GTTCAGATCA AGGTCAGGAA CAGATGGAAC AGCTGAATAT GGGCCAAACA GGATATCTGT 4730 8 GGTAAGCAGT TCCTGCCCCG GCTCAGGGCC AAGAACAGAT GGAACAGCTG AATATGGGCC 4790 AAACAGGATA TCTGTGGTAA GCAGTTCCTG CCCCGGCTCA GGGCCAAGAA CAGATGGTCC 4850 CCAGATGCGG TCCAGCCCTC AGCAGTTTCT AGAGAACCAT CAGATGTTTC CAGGGTGCCC 4910 CAAGGACCTG AAATGACCCT GTGCCTTATT TGAACTAACC AATCAGTTCG CTTCTCGCTT 4970 CTGTTCGCGC GCTTCTGCTC CCCGAGCTCA ATAAAAGAGC CCACAACCCC TCACTCGGGG 5030 CGCCAGTCCT CCGATTGACT GAGTCGCCCG GGTACCCGTG TATCCAATAA ACCCTCTTGC 5090 AGTTGCATCC GACTTGTGGT CTCGCTGTTC CTTGGGAGGG TCTCCTCTGA GTGATTGACT 5150 ACCCGTCAGC GGGGGTCTTT CATTTGG 5177 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 400 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Wing Gln Trp Asp Asp Phe Pro Asp Gln Gln Glu Asp Thr Asp Ser 1 5 10 15 Cys Thr Glu Ser Val Lys Phe Asp Ala Arg Ser Val Thr Ala Leu Leu 20 25 30 Pro Pro His Pro Lys Asn Gly Pro Thr Leu Gln Glu Arg Met Lys Ser 35 40 45 Tyr Lys Thr Ala Leu He Thr Leu Tyr Leu He Val Phe Val Val Leu 50 55 60 Val Pro He He Gly He Val Ala Wing Gln Leu Leu Lys Trp Glu Thr 65 70 75 80 Lys Asn Cys Thr Val Gly Ser Val Asn Wing Asp He Ser Pro Pro Pro 85 90 95 Glu Gly Lys Gly Asn Gly Ser Glu Asp Glu Met Arg Phe Arg Glu Ala 100 105 110 Val Met Glu Arg Met Being Asn Met Glu Being Arg He Gln Tyr Leu Ser 115 120 125 Asp Asn Glu Wing Asn Leu Leu Asp Wing Lys Aen Phe Gln Asn Phe Ser 130 135 140 He Thr Thr Asp Gln Arg Phe Asn Asp Val Leu Phe Gln Leu Asn Ser 145 150 155 160 Leu Leu Be Ser He Gln Glu His Glu Asn He He Gly Asp He. Ser 165 170 175 Lys Ser Leu Val Gly Leu Asn Thr Thr Val Leu Asp Leu Gln Phe Ser 180 185 190 He Glu Thr Leu Asn Gly Arg Val Gln Glu Asn Wing Phe Lys Gln Gln 195 200 205 Glu Glu Met Arg Lys Leu Glu Glu Arg He Tyr Asn Wing Being Glu Wing 210 215 220 He Lys Ser Leu Asp Glu Lys Gln Val Tyr Leu Glu Gln Glu He Lys 225 230 235 240 Gly Glu Met Lys Leu Leu Asn Asn He Thr Asn Asp Leu Arg Leu Lys 245 250 255 sp Tr Glu His Ser Gln Thr Leu Lys Asn He Thr Leu Leu Gln Gly 260 265 270 10 Wing Arg Lys Cys Ser Leu Thr Gly Lys Trp Thr Asn Asp Leu Gly Ser 275 280 285 Asn Met Thr He Gly Wing Val Asn Ser Arg Gly Glu Phe Thr Gly Thr 290 295 300 Tyr He Thr Wing Val Thr Wing Thr Ser Asn Glu He Lys Glu Ser Pro 305 310 315 320 Leu His Gly Thr Gln Asn Thr He Asn Lys Arg Thr Gln Pro Thr Phe 325 330 335 Gly Phe Thr Val Asn Trp Lys Phe Ser Glu Ser Thr Thr Val Phe Thr 340 345 350 Gly Gln Cys Phe He Asp Arg Asn Gly Lys Glu Val Leu Lys Thr Met 355 360 365 Trp Leu Leu Arg Ser Ser Val Asn Asp He Gly Asp Asp Trp Lys Wing 370 375 380 Thr Arg Val Gly He Asn He Phe Thr Arg Leu Arg Thr Gln Lys Glu 385 390 395 400

Claims (15)

10 CLAIMS 1. A protein comprising a membrane-spanning domain and an extracellular domain characterized in that the extracellular domain comprises biotin-binding activity.

2. The protein in accordance with the claim 1, characterized in that it additionally comprises a cytoplasmic domain.

3. The protein according to claim 1 or claim 2, characterized in that the extracellular domain comprises functional activity of avidin or streptavidin. The protein according to any of the preceding claims, characterized in that it comprises an amino acid sequence of a class A scavenger receptor. The protein according to any of claims 1 to 3, characterized in that the protein comprises a sequence of amino acids as defined in SEC. FROM IDENT. No. 2. 6. A nucleic acid molecule encoding a protein according to any of the preceding claims. 7. A recombinant expression vector comprising a nucleic acid molecule according to claim 6. 8. A process for the production of a protein of nucleic acid. according to any one of claims 1 to 5, comprising the transfection of a cell line with a recombinant expression vector according to claim 7, and the expression of the protein in the transfected cells. 9. A method for the delivery of a molecule to a target site, comprising the addition of the molecule to a solution containing the target, characterized in that the molecule is biotylated and the target comprises a protein in accordance with any of claims 1 to 5. 10. A use of a nucleic acid molecule according to claim 6, in the manufacture of a medicament for administration to a target site, wherein the protein encoded by the nucleic acid molecule is expressed in the objective site. 11. A use of a biotinylated molecule in the manufacture of a medicament for administration at a target site, wherein the target site comprises a protein according to any of claims 1 to 5. • 12. Use of a biotinylated molecule in the manufacture of a medicament for administration to a target site for treating a disease, wherein the target site comprises a protein in accordance with any of claims 1 to 5, and the biotinylated molecule exerts its effect at the target site. The protein according to claim 4, characterized in that the extracellular domain comprises avidma having a biotin binding domain. 1 . The protein according to any of claims 1 to 5, for use in therapy. 15. The nucleic acid molecule according to claim 6, for use in therapy.