WO2000055321B1 - Vertebrate protein slit, dna sequence encoding it and uses thereof - Google Patents

Abstract

Polynucleotides that encode the Xenopus slit protein along with the deduced amino acid sequence of the Xenopus slit protein are given. Recombinant polynucleotides, vectors and transformed cells containing the slit polynucleotide are disclosed. Methods for production of slit proteins, pharmaceutical compounds containing slit protein and therapeutic uses for slit protein are also given. Methods for the repulsive guidance of nerve axon growth and inhibition of cell migration using slit are provided. Methods of stimulating cell proliferation using slit are given, along with methods and compositions for reducing the use of serum in cell culture by the use of slit.

Claims

56

AMENDED CLAIMS

[received by the International Bureau on 20 February 2001 (20.02.01); original claims 10, 11 and 15 amended; remaining claims unchanged (2 pages)]

6. Λ host cell comprising the recombinant vector of claim 3.

7 The host cell of claim 6, wherein said host cell is selected from the group consisting of mammalian cells, plant cells, insect cells, ycasl, bacteria, bacteriophage.

8. The host cell of claim 6, wherein said host cell expresses a protein encoded by said vector,

9. The host cell of claim 8, wherein said expressed protein is secreted by said host cell.

10 A protein or polypcptide fragment encoded by the polynucleotide selected from the group consisting of:

(a) a polynucleotide of SEQ ID NO; 1;

(b) a polynucleotide that has at least 90% sequence identity with the polynucleotide of (a);

(c) a polynucleoluic that hybridizes to the polynucleotide of (a) under conditions of 5X SSC, 50% formamide and 42 °C, and which encodes a protein havmg the same biological function;

(d) a polynucleotide encoding the same amino acid sequence as (a), but which exhibits regular degeneracy in accordance with the degeneracy ofthe genetic code;

(e) a polynucleotide encoding the same amino acid sequence as (b), but which exhibits regular degeneracy in accordance with the degeneracy ofthe genetic code; and (f) a polynucleotide encoding the same amino acid sequence as (c), but which exhibits regular degeneracy in accordance with the degeneracy ofthe genetic code.

11. A protein comprising the amino acid sequence of SEQ ID NO: 2 or a fragment of

SEQ ID NO; 2 and having the same biological function as the protein of SEQ ID NO: 2.

MENDED SHEET (ARTICLE 19T 57

12 '[ he protein of claim 10, wherein one or more of the amino acids have been substituted with a conserved amino acid and the biological function ofthe protein has been maintained.

1 . The protein of claim 10, wherein one of more ofthe amino acid resides includes a substilucnt group.

14. A method fur the production of a puri ficd slit protein comprising growing host cells of claim 8 under conditions where said host cells express a protein encoded by said rccombmant vector and isolating said expressed protein.

15. A pharmaceutical composition comprising the protein of claim 10, a protein fragment thereof having the same biological function, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

16. A method for altering cell migration comprising administering a migration altering amount of a slit protein or a pharmaceutically acceptable salt thereof.

17. The method of claim 16 wherein said sht protein is the protein of claim 10.

18. The method of claim 16, wherein said alteration is by repulsion,

19. The method of claim 16, wherein said alteration is by inhibition,

20. The method of claim 16, wherein said cells express the roundabout (robo) protein

21. The method of claim 16, wherein said cells are neural cells.

22. The method of claim 16, wherein said cells are malignant cells.

23. The method of claim 16, wherein said cells are leukocytes.