What is claimed is:
1. A purified soluble recombinant v3 adhesion receptor which retains a ligand binding activity.
2. A soluble human v3 adhesion receptor of claim 1.
3. A receptor of claim 2, comprising the v chain and the 3 chain of said receptor, wherein each chain is shortened at its C-terminus by a portion containing the transmembrane domain or a portion thereof and the complete cytoplasmic domain.
4. A receptor of claim 3, comprising a truncated v chain containing approximately 957 amino acids and a truncated 3 chain containing approximately 692 amino acids, each starting at the N-terminal of the corresponding mature protein chain.
5. A human v3 adhesion receptor, obtainable by a process as defined in claim 7.
6. A receptor of claim 12, wherein the truncated v chain has a molecular weight of 120 kD (12 kD) and the truncated 3 chain has a molecular weight of approximately 80 kD (8 kD), each determined by SDS-PAGE under non-reducing conditions.
7. A process for preparing a large amount of a highly purified soluble recombinant human v3 adhesion receptor which retains a ligand binding activity, comprising
(i) subcloning a first cDNA coding for the v chain of said receptor, shortened by a portion comprising at least 52 amino acids calculated from the C-terminus, and a second cDNA coding for the 3 chain of said receptor, shortened by a portion comprising at least 61 amino acids calculated from the C-terminus, into a baculovirus transfer vector of a baculovirus expression system,
(ii) transferring said vector comprising said first andor second DNA into the genomic DNA of a baculovirus of said expression system,
(iii) infecting an insect cell with said complete recombinant baculovirus,
(iv) cultivating said infected insect cells in a culture medium whereby said heteromeric truncated v3 receptor is expressed into the medium, and
(v) purifying said expressed receptor from the medium by antibody affinity chromatography, wherein the antibody is specific to the human v3 adhesion receptor or its individual component chains.
8. A process of claim 7, wherein the first and second cDNA are sub-cloned into the same baculovirus vector.
9. A process of claim 7, wherein the baculovirus expression system is the BacPAK system.
10. A process of claim 7, wherein the insect cells infected are High Five (BTI-TN-5B1-4) cells.
11. A process of claim 7, wherein the specific antibody used for the antibody affinity chromatography is mAb 17E6 produced by a hybridoma cell line having the designation 272-17E6 (DSM ACC2160).
12. A process of claim 7, wherein the first cDNA codes for the v chain of said receptor, shortened by a portion encoding approximately 61 amino acids starting at the C-terminus and which corresponds to a mature protein chain containing approximately 957 amino acids, and the second cDNA codes for the 3 chain of said receptor, shortened by a portion encoding approximately 70 amino acids starting at the C-terminus and which corresponds to a mature protein chain containing approximately 692 amino acids.
13. A process of claim 12, wherein the cDNA of the truncated v chain is generated by PCR using the oligonucleotide primers
7
5-GAC CAG CAT TTA CAG TGA-3 and
5-CA CAG GTC TAG ACT ATG GCT GAA TGC CCC AGG-3,
and the cDNA of the truncated 3 chain is generated by PCR using the oligonucleotide primers 5
8
5-GCG CGC AAG CTT GCC GCC ACC ATG CGA GCG CGG CCG-3 and
5GAT CGA TCT AGA CTA GGT CAG GGC CCT TGG GAC ACT-3.
14. A method of screening a compound for its capability to inhibit an activity of a natural v3 receptor, comprising contacting a soluble v3 receptor of claim 1 with a compound which may inhibit said v3 receptor, and determining of the receptor activity is inhibited.
15. A method of claim 14, wherein the compound is an RGD-peptide or a non-peptidic analogue.
16. A method of claim 15, wherein the RGD-peptide or a non-peptidic analogue compound is modified by a detectable marker and reacted with said immobilized purified soluble v3 receptor, and the amount of ligand bound to said receptor is measured.
17. A process for preparing large amounts of a highly purified intact full-length recombinant human v3 adhesion receptor, comprising
(i) subcloning a first cDNA coding for the complete a chain of said receptor and a second cDNA coding for the 3 chain of said receptor into a baculovirus transfer vector of a baculovirus expression system,
(ii) transferring said vector comprising said first andor second DNA into the genomic DNA of a baculovirus of said expression system,
(iii) infecting insect cells with said complete recombinant baculovirus,
(iv) cultivating said infected insect cells in a culture medium, whereby said receptor is expressed into the cell memembrane,
(v) solubilizing said expressed cell membrane-bound receptor with a detergent, and
(vi) purifying said solubilized receptor from the cell-free medium by antibody affinity chromatography, wherein the antibody is specific to the human v3 adhesion receptor or its individual component chains.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A refuse bag comprising an elongate body having a closed end defining a bottom of the bag and an open end defining a mouth of the bag, wherein the body is provided with a pair of integral elongate protrusions that extend away from the body, the pair of elongate protrusions being constructed and arranged such that they can be tied together in a double knot to close the mouth of the bag, wherein each protrusion is provided with a plurality of apertures formed therein, and each protrusion has a proximal end proximate the body of the bag and an opposite distal end, and wherein each aperture is located in a portion of the respective protrusion closer to the proximal end than to the distal end of the respective protrusion, wherein after the protrusions have been tied in a double knot, the protrusions can be arranged so that the apertures in the protrusions are coincident with one another, and wherein each aperture is created so as to provide a handle and is sized to allow the digits of a hand to be inserted into the aperture.
2. A bag as claimed in claim 1, wherein each aperture is defined by a cut-out.
3. A bag as claimed in claim 1, wherein each aperture is defined by an \u201cupside-down cut-out\u201d.
4. A bag as claimed in claim 1, wherein the bag is made from a thermoplastics material such as polyethylene.
5. A roll of material defining a plurality of bags, each bag being a refuse bag in accordance with claim 1, and each bag being formed so as to be separable from the roll.
6. A roll as claimed in claim 5, wherein each aperture is defined by a cut-out.
7. A roll as claimed in claim 5, wherein each aperture is defined by an \u201cupside-down cut-out\u201d.
8. A roll as claimed in claim 5, wherein each aperture is formed by removal of material from the roll.
9. A roll as claimed in claim 5, wherein the roll of material is provided with a respective first weakened line adjacent to the closed end of each bag to enable that bag to be separated from the next bag on the roll and an open end of each bag is provided with the second weakened line defining elongate protrusions extending away from the body of that bag to enable that bag to be separated from the next bag on the roll.
10. A roll according to claim 9, wherein the first and second weakened lines are perforated lines of weakness.
11. A roll according to claim 10, wherein the perforations forming the perforated lines of weakness are of equal length.
12. A roll as claimed in claim 5, wherein the bags are arranged on the rolls such that the open end of one bag is located adjacent to the open end of an adjacent bag.
13. A roll according to claim 5, wherein the material is folded before forming a roll.
14. A roll as claimed in claim 5, wherein each aperture is defined by a cut-out portion in such a manner that the cut-out portion remains connected to the associated protrusion.
15. A bag as claimed in claim 1, wherein each aperture is defined by a cut-out portion in such a manner that the cut-out portion remains connected to the associated protrusion.