VESICULINS
The inventions relate generally to vesiculin peptides and vesiculin peptide chains, and fragments, variants and derivatives thereof, related compositions and formulations and their preparation and use, nucleic acids encoding such vesiculin peptides and vesiculin peptide chains, and fragments, variants and derivatives thereof and related vectors and host cells, hybridomas and antibodies, and methods for the prevention and treatment of conditions, diseases and disorders that would be improved, eased, or lessened by the administration of a composition of the invention, including but not limited to glucose metabolism diseases.
1 . Substantially pure vesiculin.
2 . The vesiculin of claim 1 which is a human vesiculin.
3 . The vesiculin of claim 1 which is a rat vesiculin.
4 . The vesiculin of claim 1 , wherein said vesiculin is selected from the group consisting of mouse vesiculins, horse vesiculins, cow vesiculins, sheep vesiculins, and chicken vesiculins.
5 . The vesiculin of claim 1 , wherein said vesiculin is selected from the group consisting of salmon vesiculins, eel vesiculins, bream vesiculins, and shark vesiculins.
6 . The vesiculin of claim 1 , wherein said vesiculin is a kangaroo vesiculin.
7 . The vesiculin of claim 1 wherein said vesiculin has 62 amino acids.
8 . The vesiculin of claim 1 wherein said vesiculin has 63 amino acids.
9 . The vesiculin of claim 1 wherein said vesiculin is at least about 90% pure.
10 . The vesiculin of claim 2 , wherein said vesiculin is at least about 95% pure.
11 . An isolated polynucleotide encoding a vesiculin.
12 . The isolated polynucleotide of claim 11 , wherein said vesiculin is a human vesiculin.
13 . The isolated polynucleotide of claim 11 , wherein said vesiculin is a rat vesiculin.
14 . The isolated polynucleotide of claim 11 , wherein said vesiculin is selected from the group consisting of mouse vesiculins, horse vesiculins, cow vesiculins, sheep vesiculins, kangaroo vesiculins, and chicken vesiculins.
15 . The isolated polynucleotide of claim 11 , wherein said vesiculin is selected from the group consisting of salmon vesiculins, eel vesiculins, bream vesiculins, and shark vesiculins.
16 . An expression vector comprising a polynucleotide according to claim 11 .
17 . A recombinant host cell comprising the expression vector of claim 16 .
18 . The recombinant host cell of claim 17 , wherein said host cell is mammalian.
19 . The host cell of claim 18 , wherein said mammalian cell is a CHO cell.
20 . The recombinant host cell of claim 17 , wherein said host cell is a bacterial.
21 . The host cell of claim 20 , wherein said bacterial cell is E. coli.
22 . An isolated polypeptide comprising a first chain having the amino acid sequence:
Gly Ile Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu Ala Leu Leu Glu Thr Tyr
Cys 6 Ala R 7 Pro Ala Lys Ser Glu
and a second chain having the amino acid sequence:
Ala 1 Tyr R 1 Pro R 2 Glu Thr Leu Cys 1 Gly Gly Glu Leu Val Asp Tyr Leu Gln Phe
Val Cys 2 R 3 Asp Arg Gly Phe Tyr Phe Ser Arg Pro R 4 Ser Arg R 5 R 6
wherein Ala 1 is either present or absent; R 1 is Gly or Arg, or a conservative variant of either; R 2 is Gly or Ser, or a conservative variant of either; R 3 is Gly or Ser, or a conservative variant of either; R 4 is Ser or Ala, or a conservative variant of either; R 5 is Ile or Val or Ala, or a conservative variant of either; R 6 is Asn or Ser, or a conservative variant of either; and R 7 is Thr or Ala, or a conservative variant of either.
23 . An isolated polypeptide comprising a first chain having the amino acid sequence
Gly Ile Val Glu Glu Cys 3 Cys 4 Phe Arg Ser Cys 5 Asp Leu R 1 R 2 Leu Glu R 3 Tyr
Cys 6 Ala R 4 R 5 R 6 R 7 R 8 R 9
and a second chain having the amino acid sequence:
R 10 R 11 R 12 R 13 R 14 Glu Thr Leu Cys 1 Gly Gly Glu Leu Val Asp R 15 Leu Gln Phe
R 16 Cys 2 R 17 R 18 Arg Gly Phe Tyr Phe R 19 R 20 R 21 R 22 R 23 R 24 R 25 R 26 R 27 R 28 R 29
wherein R 1 is Ala, Asn, Leu, or a conservative variant of either, R 2 is Leu, Ile, or a conservative variant of either, R 3 is Thr, Gln, or a conservative variant of either, R 4 is Thr, Ala, Lys, Val, or a conservative variant of either, R 5 is Pro, Ser, or a conservative variant of either, R 6 is Ala, Val, Pro, or a conservative variant thereof, R 7 is Lys, Glu, or a conservative variant of either, R 8 is Ser, Ala, or a conservative variant of either, R 9 is Glu, Ala, or a conservative variant of either, R 10 is absent or is Ala, Glu, Asp, or a conservative variant thereof, R 11 is Tyr, Ala, Val, or a conservative variant of either, R 12 is Arg, Gly, Ala, or a conservative variant thereof, R 13 is Pro, Thr, Ser, Leu, or a conservative variant thereof, R 14 is Ser, Gly, Ala, Glu, or a conservative variant thereof, R 15 is Thr, Tyr, Ala, or a conservative variant thereof, R 16 is Val, Ile, or a conservative variant thereof, R 17 is Gly, Ser, Glu, Ala, or a conservative variant thereof, R 18 is Asp, Glu, or a conservative variant of either, R 19 is Ser, Val, or a conservative variant of either, R 20 is Arg, Leu, Ser, or a conservative variant thereof, R 21 is Pro, Lys, or a conservative variant of either, R 22 is Ala, Ser, Gly, Val, Thr, or a conservative variant thereof, R 23 is Ser, Gly, Val, or a conservative variant thereof, R 24 is Arg, Pro, Gly, or a conservative variant thereof, R 25 is Ala, Arg, Val, Ile, Leu, Asn, Ser, Gly, or a conservative variant thereof, R 26 is Ser, Asn, Arg, or a conservative variant thereof, R 27 is absent or is Arg, Ser, Asn, or a conservative variant thereof, R 28 is absent or is Val, or a conservative variant thereof, R 29 is absent or is Ser, or a conservative variant thereof.
24 . An isolated polypeptide of claim 23 , wherein the first and second chains are joined by one or more disulfide bonds.
25 . An isolated polypeptide of claim 24 , wherein the first and second chains are joined by two disulfide bonds.
26 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 1 .
27 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 2 .
28 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 23 .
29 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 24 .
30 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 25 .
31 . A method of producing a recombinant vesiculin having an A chain and a B chain comprising of the steps of: i) preparing a polynucleotide sequence having the sequence vesiculin B chain-Asn-Gly-vesiculin A chain; ii) introducing said polynucleotide sequence into a vector suitable for expression in yeast; iii) transfecting the vector into a yeast and expressing said vector therein; iv) isolating a vesiculin B chain-Asn-Gly-vesiculin A chain polypeptide; and v) removing the Asn-Gly linker and forming said vesiculin.
32 . A method according to claim 31 , wherein said vector is a pRS400 or pESC expression vector.
33 . A method of synthesizing vesiculin having an A chain and a B chain comprising of the steps of: i) synthesizing a vesiculin A chain by solid phase or solution phase synthesis, or by using variations thereof; ii) synthesizing a vesiculin B chain solid phase or solution phase synthesis; iii) bringing said vesiculin A chain and vesiculin B chain together under conditions favoring the formation of disulfide bonds