Compositions and methods for improving production of recombinant polypeptides

Abstract

The present invention relates to biologically active polypeptides linked to one or more accessory polypeptides. 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 three types of amino acid residues, whereby said random coil conformation mediates an increased in vivo and 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 consists of alanine, serine and proline residues. 
     
     
         3 . 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. 
     
     
         4 . The biologically active protein according to  claim 2 , wherein said proline residues constitute more than 4% and less than 40% of the amino acids of said second domain forming random coil conformation. 
     
     
         5 . 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. 
     
     
         6 . The biologically active protein according to  claim 1 , wherein said polypeptide with biological activity is selected from the group consisting of binding molecules, antibody fragments, cytokines, growth factors, hormones or 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), domain antibodies and lipocalins. 
     
     
         8 . The biologically active protein according to  claim 1 , wherein said polypeptide 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, gp120/gp160, soluble tumor necrosis factor I and II receptor, 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 comprising said random coil forming second domain when compared to said biologically active protein lacking said random coil forming second domain. 
     
     
         10 . A composition comprising the biologically active protein according to any one of  claims 1  to  9 . 
     
     
         11 . The composition according to  claim 10 , which is a pharmaceutical composition, optionally further comprising a pharmaceutical acceptable carrier. 
     
     
         12 . An isolated nucleic acid molecule encoding the biologically active protein of any one of  claims 1  to  9 . 
     
     
         13 . A vector comprising the nucleic acid of  claim 12 . 
     
     
         14 . An isolated cell comprising the nucleic acid according to  claim 12 . 
     
     
         15 . A method for the preparing a biologically active protein comprising culturing the cell according to  claim 14  and isolating said biologically active protein from the culture. 
     
     
         16 . 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 11 .