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. An unstructured recombinant polymer (URPs), wherein the URP comprising at least about 200 contiguous amino acids, and 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 80% of the total amino acids of the URP;
(b) at least 50% of the amino acids of the URP are not present in secondary structure as determined by Chou-Fasman algorithm;
(c) the URP has a Tepitope score equal to or less than −5;
(d) the URP contains only 3, 4, 5, or 6 different types of amino acids; and
(e) the URP contains greater than 5% glutamic acid and less than 2% lysine or arginine;
wherein incorporation of the URP into a heterologous protein results in a fusion protein having at least a 2-fold increase in apparent molecular weight of the fusion protein as compared to the protein lacking said URP wherein the apparent molecular weight is approximated by size exclusion chromatography, and wherein the fusion protein exhibits an increased half-life as compared to the protein lacking said URP.
2. The URP of claim 1 , wherein upon incorporation of the URP into an N-terminus, a C-terminus, into a loop of the heterologous protein, or between domains of the heterologous protein to create a monomeric fusion protein, said heterologous protein exhibits a serum secretion half-life that is at least two-fold longer as compared to the corresponding protein that is deficient in said URP.
3. The URP of claim 1 , wherein the URP comprises a non-natural amino acid sequence.
4. The URP of claim 1 , wherein the URP comprises at least 400 contiguous amino acids.
5. The URP of claim 1 , wherein the URP comprises at least five 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).
6. The URP of claim 1 , wherein the URP comprises at least six 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).
7. The URP of claim 1 , wherein one type of the amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) constitutes at least 20% of the total amino acids of the URP.
8. The URP of claim 7 , wherein the one type of amino acid is glutamate (E).
9. The URP of claim 1 , wherein any one type of amino acid is no more than 30% of the total amino acids of the URP.
10. The URP of claim 1 , wherein the heterologous protein is a pharmaceutically active protein.
11. The URP of claim 1 , wherein the heterologous protein comprises one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.
12. The URP of claim 10 , wherein the pharmaceutically active 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, methioninase.
13. The URP of claim 1 , wherein any amino acids of the URP that are not glycine, aspartate, alanine, serine, threonine, glutamate, or proline are not hydrophobic amino acids selected from the group consisting of tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.