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1. WO2001072768 - COMPLEXES MAJEURS D'HISTOCOMPATIBILITE MONOCATENAIRES DE CLASSE 1, CONSTRUCTIONS LES CODANT ET LEURS METHODES DE PRODUCTION

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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WHAT IS CLAIMED IS:

1. A nucleic acid construct comprising a first nucleic acid sequence including a first polynucleotide encoding a functional human β-2 microglobulin, being translationaly fused upstream of a second polynucleotide encoding a functional human MHC class I heavy chain.

2. The nucleic acid construct of claim 1, wherein said first nucleic acid sequence further includes an in-frame linker polynucleotide encoding a linker peptide interposed between said first and said second polynucleotides.

3. The nucleic acid construct of claim 2, wherein said linker peptide is as set forth in SEQ ID NO: 10.

4. The nucleic acid construct of claim 1, wherein said first nucleic acid sequence further includes an in-frame tag sequence encoding a peptide capable of being enzymatically modified to include a binding entity.

5. The nucleic acid construct of claim 1, further comprising a second nucleic acid sequence encoding an antigenic peptide. said antigenic peptide being capable of binding a human MHC class I complex.

6. The nucleic acid construct of claim 1 , wherein said second polynucleotide encodes α 1-3 domains of said human MHC class I heavy chain.

7. The nucleic acid construct of claim 2, further comprising a first cis acting regulatory sequence for regulating expression of said first nucleic acid sequence.

8. The nucleic acid construct of claim 5, further comprising a second cis acting regulatory sequence for regulating expression of said second nucleic acid sequence.

9. The nucleic acid construct of claim 7, wherein said cis acting regulatory sequence is functional in a bacterial host.

10. A nucleic acid construct comprising a nucleic acid sequence as set forth in SEQ ID NO:4.

1 1. A transformed cell comprising the nucleic acid construct of claim 1.

12. The transformed cell of claim 11, wherein the cell is a eukaryotic cell selected from the group consisting of a mammalian cell, an insect cell, a plant cell, a yeast cell and a protozoa cell.

13. The transformed cell of claim 11, wherein the cell is a bacterial cell.

14. A transformed cell comprising the nucleic acid construct of claim 5.

15. The transformed cell of claim 14, wherein the cell is a eukaryotic cell selected from the group consisting of a mammalian cell, an insect cell, a plant cell, a yeast cell and a protozoa cell.

16. The transformed cell of claim 14. wherein the cell is a bacterial cell.

17. A recombinant polypeptide comprising an amino acid sequence including a functional human β-2 microglobulin directly or indirectly covalently linked to a functional human MHC class I heavy chain.

18. The recombinant polypeptide of claim 17, further comprising a linker peptide being interposed between said functional human β-2 microglobulin and said functional human MHC class I heavy chain.

19. A recombinant polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 5

20. A preparation of bacterial derived inclusion bodies comprising over 30 percent by weight of a recombinant polypeptide including an amino acid sequence including a functional mammalian β-2 microglobulin directly or indirectly covalently linked to a functional mammalian MHC class I heavy chain.

21. A host cell being co-transformed with:
(a) a first expression construct including a first polynucleotide encoding a functional mammalian β-2 microglobulin, being translationally fused upstream of a second polynucleotide encoding a functional MHC class I heavy chain; and
(b) a second expression construct including a third polynucleotide encoding an antigenic peptide, wherein when said first, second and third polynucleotides are co- expressed in the host cell, an MHC class I-antigenic peptide complex is formed.

22. The host cell of claim 21. wherein said first expression construct further includes an in-frame linker polynucleotide sequence encoding a linker peptide interposed between said first and said second polynucleotides.

23. The host cell of claim 21, w herein said cell is a eukaryotic cell.

24. The host cell of claim 21. wherein said cell is a bacterial cell.

25. A nucleic acid construct comprising:
(a) a first nucleic acid sequence including:
(i) a first polynucleotide encoding a functional
mammalian β-2 microglobulin; and
(ii) a second polynucleotide encoding a functional
mammalian MHC class I heavy chain, said second
polynucleotide being translationally fused
downstream of said first polynucleotide; and
(b) a cis acting regulatory sequence being selected capable for directing expression of said first nucleic acid sequence in bacteria.

26. The nucleic acid construct of claim 25, wherein said cis acting regulatory sequence is selected from the group consisting of a bacterial derived cis acting regulatory sequence and a phage derived cis acting regulatory sequence.

27. The nucleic acid construct of claim 25, wherein said first nucleic acid sequence further includes an in-frame linker polynucleotide encoding a linker peptide interposed between said first and said second polynucleotides.

28. The nucleic acid construct of claim 26, wherein said linker peptide is as set forth in SEQ ID NO: 10.

29. The nucleic acid construct of claim 25, further comprising a second nucleic acid sequence encoding an antigenic peptide. said antigenic peptide being capable of binding a mammalian MHC class I complex.

30. The nucleic acid construct of claim 25, wherein said second polynucleotide encodes α 1-3 domains of said mammalian MHC class I heavy chain.

31. A transformed cell comprising the nucleic acid construct of claim 25.

32. The transformed cell of claim 31, wherein the cell is a eukaryotic cell.

33. The transformed cell of claim 31, wherein the cell is a bacterial cell.

34. A transformed cell comprising the nucleic acid construct of claim 27.

35. A method of producing a functional MHC class I molecule comprising the steps of:
(a) expressing, in bacteria, a single chain MHC class I polypeptide including a functional mammalian β-2 microglobulin amino acid sequence directly or indirectly covalently linked to a functional mammalian MHC class I heavy chain amino acid sequence; and
(b) isolating said single chain MHC class I polypeptide.

36. The method of claim 35, further comprising the step of:
(c) refolding said single chain MHC class I polypeptide in presence of an antigenic peptide capable of binding said single chain MHC class I polypeptide, to thereby generate an MHC class I-antigenic peptide complex.

37. The method of claim 35, further comprising the step of:
(d) isolating said MHC class I-antigenic peptide complex via size exclusion chromatography.

38. The method of claim 35, wherein said antigenic peptide is co-expressed along with said single chain MHC class I polypeptide in said bacteria.

39. The method of claim 35, wherein step (a) is effected such that said single chain MHC class I polypeptide forms inclusion bodies in said bacteria.

40. The method of claim 38, wherein said antigenic peptide and said single chain MHC class I polypeptide form inclusion bodies in said bacteria.

41. The method of claim 39, wherein said step of isolating said polypeptide further includes the steps of:
(i) denaturing said inclusion bodies so as to release protein molecules therefrom; and
(ii) renaturing said protein molecules.

42. The method of claim 41, wherein said step of renaturing said protein molecules is effected in the presence of an antigenic peptide capable of binding said single chain MHC class I polypeptide.

43. The method of claim 42, wherein said antigenic peptide is co-expressed along with said single chain MHC class I polypeptide in said bacteria.

44. The method of claim 35, wherein said mammalian β-2 microglobulin amino acid sequence is a human β-2 microglobulin amino acid sequence and further wherein said mammalian MHC class I heavy chain amino acid sequence is a human MHC class I heavy chain amino acid sequence.

45. A multimeric MHC class I complex comprising a plurality of recombinant polypeptide monomers each including a functional human β-2 microglobulin directly or indirectly covalently linked to a functional human MHC class I heavy chain.

46. The multimeric MHC class I complex of claim 45, wherein said plurality of recombinant polypeptide monomers are linked to a common substrate.