Unstructured recombinant polymers and uses thereof

Abstract

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 . An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein:
 (a) the URP comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues, wherein the sum of said group of amino acids contained in the URP constitutes more than about 80% of the total amino acids of the URP, and wherein said URP comprises more than one proline residue, and wherein said URP possesses reduced sensitivity to proteolytic degradation relative to a corresponding URP lacking said more than one proline residue;   (b) at least 50% of the amino acids of said URP are devoid of secondary structure as determined by Chou-Fasman algorithm; and   (c) the Tepitope score of said URP is less than −5.   
     
     
         2 . The URP of  claim 1 , wherein more than about 20% of the total amino acids in said URP are proline (P) residues. 
     
     
         3 . An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein:
 (a) the URP comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues, wherein the sum of said group of amino acids contained in the URP, constitutes more than about 80% of the total amino acids of the URP, and wherein said URP comprises at least one other type of amino acid selected from the group consisting of cysteine (C) and lysine (K);   (b) at least 50% of the amino acids of said URP are devoid of secondary structure as determined by Chou-Fasman algorithm; and   (c) the Tepitope score of said URP is less than −5.   
     
     
         4 . An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein:
 (a) the URP comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues, wherein the sum of said at least three different types of amino acids constitutes more than about 80% of the total amino acids of the URP, and wherein said URP comprises more than about 5% glutamate (E) residues;   (b) at least 50% of the amino acids of said URP are devoid of secondary structure as determined by Chou-Fasman algorithm; and   (c) the Tepitope score of said URP is less than −5.   
     
     
         5 . An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein
 (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP constitutes more than about 90% of the total amino acids of the URP, wherein said URP does not comprise methionine (M) and/or cysteine (C) residues;   (b) the URP comprises at least three different types of amino acids;   (c) at least 50% of the amino acids of said URP are devoid of secondary structure as determined by Chou-Fasman algorithm; and   (d) the Tepitope score of said URP is less than −5.   
     
     
         6 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein upon incorporation of the URP into a heterologous protein, said heterologous protein exhibits a serum secretion half-life that is at least two times longer as compared to a corresponding protein lacking said URP. 
     
     
         7 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein incorporation of the URP into a heterologous protein results in at least a 2-fold increase in apparent molecular weight of the protein as compared to a corresponding protein lacking said URP, as approximated by size exclusion chromatography. 
     
     
         8 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein said at least 40 contiguous amino acids are predominantly hydrophilic residues. 
     
     
         9 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein at least 15% of the amino acids of the URP are glutamic acid and less than 2% of the amino acids are lysine or arginine. 
     
     
         10 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein any one type of the amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), and glutamate (E) constitutes more than about 20% of the total amino acids of the URP. 
     
     
         11 . The URP of any one of  claims 1 ,  3 ,  4  and  5 , wherein the URP comprises more than about 100 contiguous amino acids. 
     
     
         12 . The URP of  claim 11 , wherein the URP comprises more than about 200 contiguous amino acids. 
     
     
         13 . A protein comprising one or more URPs of any one of  claims 1 ,  3 ,  4  and  5 , wherein said one or more URPs are heterologous with respect to the protein. 
     
     
         14 . The protein of  claim 13 , comprising one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules. 
     
     
         15 . The protein of  claim 13 , wherein the protein is selected from the group consisting of cytokines, growth factors, enzymes, receptors, microproteins, hormones, erythopoetin, adenosine deiminase, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, rasburicase, axokine, RNAse, DNAse, phosphatase, pseudomonas exotoxin, ricin, gelonin, desmoteplase, laronidase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor VII, factor VIII, factor X, factor IX, dornase, glucocerebrosidase, follitropin, glucagon, thyrotropin, nesiritide, alteplase, teriparatide, agalsidase, laronidase, and methioninase. 
     
     
         16 . A recombinant polynucleotide comprising a coding sequence that encodes the URP of any one of  claims 1 ,  3 ,  4  and  5 . 
     
     
         17 . A vector comprising the recombinant polynucleotide of  claim 16 . 
     
     
         18 . A host cell comprising the recombinant polynucleotide of  claim 16 .