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 - 148 . (canceled) 
     
     
         149 . 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 biological activity; and (b) a second domain of said at least two domains consisting of at least about 80 amino acid residues consisting essentially of alanine, serine and proline residues, wherein said second domain substantially lacks secondary structure and mediates an increased in vivo and in vitro stability of said biologically active protein compared to said biologically active protein lacking said second domain. 
     
     
         150 . The biologically active protein according to  claim 149 , wherein the second domain substantially lacks secondary structures of alpha helices or beta-sheets. 
     
     
         151 . The biologically active protein according to  claim 149 , wherein the second domain forms a random coil conformation. 
     
     
         152 . The biologically active protein according to  claim 149 , wherein said second domain mediates an increase in serum half-life of said biologically active protein. 
     
     
         153 . The biologically active protein according to  claim 149 , wherein said second domain comprises a plurality of amino acid repeats, and wherein no more than 6 consecutive amino acid residues are identical. 
     
     
         154 . The biologically active protein according to  claim 149 , wherein said proline residues constitute more than 4% and less than 40% of the amino acids of said second domain. 
     
     
         155 . The biologically active protein according to  claim 149 , wherein said second domain comprises an amino acid sequence consisting of about 100 to 3000 amino acid residues. 
     
     
         156 . The biologically active protein according to  claim 149 , wherein said first domain with biological activity is selected from the group consisting of binding molecules, antibody fragments, cytokines, growth factors, hormones and enzymes. 
     
     
         157 . The biologically active protein according to  claim 156 , 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. 
     
     
         158 . The biologically active protein according to  claim 149 , wherein said first domain comprising an amino acid sequence having biological activity is selected from the group consisting of human growth hormone (hGH), glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), interferon-alpha, interferon-beta, interferon-gamma, insulin, erythropoietin, tumor necrosis factor (TNF), coagulation factor VIII, gp120, gp160, soluble tumor necrosis factor I and II receptor, reteplase, exendin-4, anakinra, interleukin-2, neutrophil gelatinase-associated lipocalin, tumor necrosis factor-alpha (TFN-alpha), IL-1RA, exenatide, uricase and pramlitide. 
     
     
         159 . The biologically active protein according to  claim 149 , wherein said second domain consists of no more than 5% of residues other than alanine, serine and proline. 
     
     
         160 . The biologically active protein according to  claim 149 , wherein said residues other than alanine, serine and proline are selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. 
     
     
         161 . A composition comprising the biologically active protein according to  claim 149 . 
     
     
         162 . The composition according to  claim 161 , which is a pharmaceutical composition, optionally further comprising a pharmaceutical acceptable carrier. 
     
     
         163 . A nucleic acid molecule encoding the biologically active protein of  claim 149 . 
     
     
         164 . A vector comprising the nucleic acid of  claim 163 . 
     
     
         165 . A host cell comprising the nucleic acid according to  claim 163 . 
     
     
         166 . A method for the preparation of the biologically active protein according to  claim 149  comprising culturing a cell comprising (a) a nucleic acid molecule encoding the biologically active protein of  claim 149  or (b) a vector comprising a nucleic acid molecule encoding the biologically active protein of  claim 149  and isolating said biologically active protein from the culture. 
     
     
         167 . A method of treating hormone deficiency-related disorders, auto-immune disease, cancer, anemia, vascular diseases, infectious/inflammatory diseases, thrombosis, myocardial infarction, diabetes, hepatorenal syndrome or other kidney diseases comprising administering the biologically active protein according to  claim 149 , a nucleic acid encoding the biologically active protein of  claim 149 , a vector comprising a nucleic acid and encoding the biologically active protein of  claim 149 , or a cell comprising (a) a nucleic acid molecule encoding the biologically active protein of  claim 149  or (b) a vector comprising a nucleic acid molecule encoding the biologically active protein of  claim 149 .