BIOLOGICALLY ACTIVE PROTEINS HAVING INCREASED IN VIVO AND/OR IN VITRO STABILITY
The present invention provides unstructured recombinant polymers (URPs) and proteins containing one or more of the URPs. The present invention also provides microproteins, toxins and other related proteinaceous entities, as well as genetic packages displaying these entities. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.
1 . A biologically active protein comprising at least two domains wherein (a) a first domain of said at least two domains comprises an amino acid sequence having and/or mediating said biological activity; and (b) a second domain of said at least two domains comprises an amino acid sequence consisting of at least about 100 amino acid residues forming random coil conformation, wherein said second domains consists of alanine, serine and proline residues whereby said random coil conformation mediates an increased in vivo and/or in vitro stability of said biologically active protein.
2 . The biologically active protein according to claim 1 , wherein said second domain forming random coil conformation comprises a plurality of amino acid repeats, wherein said repeat consist of Ala, Ser, and Pro residues and wherein no more than 6 consecutive amino acid residues are identical.
3 . The biologically active protein according to claim 1 , wherein said proline residues constitute more than 4% and less than 40% of the amino acids of said second domain forming random coil conformation.
4 . The biologically active protein according to claim 1 , wherein said second domain of said at least two domains comprises an amino acid sequence consisting of about 100 to 3000 amino acid residues forming random coil conformation.
5 . The biologically active protein according to claim 1 , wherein said polypeptide with biological activity is selected from the group consisting of binding modules, antibody fragments, cytokines, growth factors, hormones or enzymes.
6 . The biologically active protein according to claim 5 wherein said binding modules is selected from the group consisting of antibodies, antibody fragments, domain antibodies and lipocalins.
7 . The biologically active protein according to claim 1 , wherein said domain with 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, soluble tumor necrosis factor I and II receptor, interleukin 2 and neutrophil gelatinase associated lipocalin.
8 . The biologically active protein according to claim 50 , wherein said increased in vivo stability of said biologically active protein is a prolonged plasma half life of said biologically active protein comprising said random coil forming second domain when compared to said biologically active protein lacking said random coil forming second domain.
9 . A composition comprising the biologically active protein according to any one of claims 1 - 8 , optionally further comprising a pharmaceutical acceptable carrier.
10 . A nucleic acid molecule encoding the biologically active protein of any one of claims 1 - 8 .
11 . A vector comprising the nucleic acid of claim 10 .
12 . A cell comprising the nucleic acid according to claim 10 .
13 . A method for preparing a biologically active protein comprising culturing the cell according to claim 12 and isolating said biologically active protein from the culture.
14 . A method of treating a disease condition selected from the group consisting of hormone deficiency related disorders, autoimmune disease, cancer, anaemia, neovascular diseases, infectious/inflammatory diseases, thrombosis, myocardial infarction, diabetes, reperfusion injury, and a kidney disease, comprising administering to a subject in need thereof a composition according to claim 9 .