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Details for Patent: 5,670,341

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Details for Patent: 5,670,341

Title: DNA vectors, hosts, probes and recombinant methods of preparing human somatomedin-like carrier proteins or polypeptides
Abstract:Methods and compositions are disclosed for producing human somatomedin carrier protein-like polypeptides. Human somatomedin carrier protein-like polypeptides, which bind somatomedins, are produced using recombinant technology. Also disclosed are methods for making complexes of human somatomedin carrier protein-like polypeptides and somatomedins.
Inventor(s): Spencer; Emerald Martin (San Francisco, CA), Talkington-Verser; Carol (San Rafael, CA)
Assignee: Celtrix Pharmaceuticals, Inc. (Santa Clara, CA)
Filing Date:May 19, 1995
Application Number:08/444,419
Claims:1. A recombinant DNA molecule comprising: a first DNA sequence encoding amino acids 27-290 of FIG. 4, wherein said first DNA sequence is preceded by a second DNA sequence encoding a secretion or signal sequence not normally found in a gram-negative bacterial host cell, such that translation is initiated at the beginning of or upstream of said second DNA wherein said second DNA is operably linked to an expression control sequence of a bacterial origin, and wherein the polypeptide encoded by said sequence accumulates inside said gram-negative bacterial host cell in soluble form.

2. A gram-negative bacterial host transformed with at least one recombinant DNA molecule of claim 1.

3. A vector comprising a recombinant DNA molecule according to claim 1.

4. A gram-negative bacterial host transformed with a recombinant DNA molecule of claim 1 and with a recombinant DNA molecule consisting essentially of a DNA molecule which codes for a carrier-protein sub-unit operatively linked to an expression control sequence.

5. The DNA of claim 1 wherein said gram-negative bacterial host cell is E. coli.

6. A method for producing a carrier protein-like polypeptide comprising

(a) transforming a gram-negative bacterial host with at least one recombinant DNA molecule of claim 1 and

(b) culturing such host to produce said polypeptide.

7. A method for producing a composition comprising at least one polypeptide comprising amino acids 27-290 of FIG. 4 substantially complexed with at least one somatomedin-like polypeptide, said method comprising: transforming a gram-negative bacterial host with at least one recombinant DNA molecule of claim 1; co-transforming said host with at least one DNA molecule which codes for a somatomedin operatively linked to an expression control sequence; and culturing said host to produce said polypeptides, wherein said polypeptide comprising amino acids 27-290 of FIG. 4 is completely free of glycosylation.

8. The method of claim 7 wherein said gram-negative bacterial host cell is E. coli

9. A gram-negative bacterial host transformed with a vector according to claim 3.

10. A method of claim 6, comprising the additional step of collecting the polypeptide.

11. A recombinant DNA molecule comprising: a DNA construct, said DNA construct comprising a first DNA sequence encoding amino acids 27-290 of FIG. 4 and a second DNA sequence encoding a signal sequence from preproinsulin, said second DNA sequence preceding said first DNA sequence and said second DNA sequence fused in frame to said first DNA sequence; and an expression control sequence of bacterial origin, wherein said expression control sequence is operably linked to said DNA construct and wherein expression of said DNA construct in a host cell results in the accumulation of a soluble protein within said host cell, said soluble protein comprising the amino acid sequence of amino acids 27-290 of FIG. 4.

12. A vector comprising a recombinant DNA molecule according to claim 11.

13. A host cell transformed with at least one recombinant DNA molecule accordingly to claim 11.

14. A host cell transformed with a vector according to claim 12.

15. A method of producing a recombinant protein comprising the amino acid sequence of amino acids 27-290 of FIG. 4, said method comprising culturing a host cell according to claim 14 under conditions appropriate for the accumulation of said polypeptide.

16. The method of claim 15 further comprising collecting said polypeptide from said host cells.

17. The method of claim 15, wherein said polypeptide is completely free of glycosylation.

18. The DNA of claim 11 wherein said host cell is bacterial.

19. The DNA of claim 18 wherein said bacterial host cell is gram-negative.

20. The DNA of claim 19 wherein said gram-negative bacterial host cell is E. coli.
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