Biological active proteins having increased in vivo and/or in vitro stability
Increased in vivo and/or in vitro stability is imparted to a biologically active protein by fusing to an amino acid sequence consisting of at least about 100 amino acid residues, which are Alanine, Serine and Proline, which form a random coil conformation. Specific examples are described. Also described are related nucleic acids, vectors and cells encoding such amino acids; compositions of biologically active proteins fused to a random coil domain, and methods of making and using the compounds and compositions of the invention.
1. A biologically active protein comprising
(a) a first domain comprising an amino acid sequence having said biological activity; and
(b) a second domain consisting of at least 80 amino acid residues consisting of alanine, serine and proline residues, and which forms a random coil conformation which mediates an increased in vivo and/or in vitro stability of said biologically active protein compared with the biologically active protein lacking the second domain.
2. The biologically active protein according to claim 1 , wherein said second domain comprises a plurality of amino acid repeats and wherein no more than 6 consecutive amino acid residues are the same amino acid.
3. The biologically active protein according to claim 1 , wherein said proline residues consist of 4% to 40% of said second domain.
4. The biologically active protein according to claim 1 , wherein said second domain comprises the amino acid sequence selected from the group consisting of
ASPAAPAPASPAAPAPSAPA;
(SEQ ID NO: 18)
AAPASPAPAAPSAPAPAAPS;
(SEQ ID NO: 20)
APSSPSPSAPSSPSPASPSS;
(SEQ ID NO: 22)
SAPSSPSPSAPSSPSPASPS;
(SEQ ID NO: 63)
SSPSAPSPSSPASPSPSSPA;
(SEQ ID NO: 24)
AASPAAPSAPPAAASPAAPSAPPA;
(SEQ ID NO: 26)
and
ASAAAPAAASAAASAPSAAA.
(SEQ ID NO: 28)
.
5. The biologically active protein according to claim 1 , wherein said second domain consists of about 100 to 3000 amino acid residues.
6. The biologically active protein according to claim 1 , wherein said first domain is selected from the group consisting of binding molecules, antibody fragments, cytokines, growth factors, hormones and enzymes.
7. The biologically active protein according to claim 6 , wherein said binding molecule is selected from the group consisting of antibodies, Fab fragments, F(ab′) 2 fragments, CDR derived peptidomimetics, single chain variable fragments (scFv), lectins and lipocalins.
8. The biologically active protein according to claim 1 , wherein said first domain comprising an amino acid sequence having biological activity is selected from the group consisting of granulocyte colony stimulating factor, human growth hormone, alpha-interferon, beta-interferon, gamma-interferon, tumor necrosis factor, erythropoietin, coagulation factor VIII, gp120/gp160, soluble tumor necrosis factor I and II receptor, reteplase, exendin-4, anakinra, interleukin-2, and neutrophil gelatinase-associated lipocalin.
9. The biologically active protein according to claim 1 , wherein said increased in vivo stability of said biologically active protein is a prolonged plasma half-life of said biologically active protein.
10. A composition comprising the biologically active protein according to claim 1 .
11. The composition according to claim 10 which is a pharmaceutical composition, optionally further comprising a pharmaceutical acceptable carrier.
12. A nucleic acid molecule encoding the biologically active protein of claim 1 .
13. A vector comprising the nucleic acid of claim 12 .
14. A cell comprising the nucleic acid according to claim 12 .
15. A method for the preparation of the biologically active protein according to claim 1 comprising culturing a cell comprising (a) a nucleic acid molecule encoding the biologically active protein of claim 1 or (b) a vector comprising a nucleic acid molecule encoding the biologically active protein of claim 1 and isolating said biologically active protein from the culture.
16. Kit comprising the biologically active protein according to claim 1 , a nucleic acid encoding the biologically active protein of claim 1 , a vector comprising a nucleic acid and encoding the biologically active protein of claim 1 or a cell comprising (a) a nucleic acid molecule encoding the biologically active protein of claim 1 or (b) a vector comprising a nucleic acid molecule encoding the biologically active protein of claim 1 .
17. A method of treating hormone deficiency-related disorders, auto-immune disease, cancer, anemia, neovascular diseases, infectious/inflammatory diseases, thrombosis, myocardial infarction, diabetes, reperfusion injury or other kidney diseases comprising administering the biologically active protein according to claim 1 , a nucleic acid encoding the biologically active protein of claim 1 , a vector comprising a nucleic acid and encoding the biologically active protein of claim 1 or a cell comprising (a) a nucleic acid molecule encoding the biologically active protein of claim 1 or (b) a vector comprising a nucleic acid molecule encoding the biologically active protein of claim 1 .