Salmon follitropin hormone analogs
View Patent ↗This invention relates to the field of glycoprotein hormone analogs and their uses as agonists, antagonists, targeting vectors, and immunogens. In particular, this invention describes a method for stabilizing a heterodimer that permits the preparation of functional glycoprotein hormone analogs. The analogs of present invention comprise at least one alpha subunit polypeptide and at least one beta subunit polypeptide, wherein the seatbelt region of the beta subunit is linked to the alpha subunit. The invention also provides for a beta subunit polypeptide wherein the C-terminal amino acid is from residue 10 to residue 20 of the seatbelt region.
1. A biologically active salmon follitropin glycoprotein hormone analog, capable of binding to a follicle stimulating hormone receptor, said analog consisting essentially of a heterodimer comprising a glycosylated α subunit polypeptide and a glycosylated β subunit polypeptide, wherein:
said α subunit polypeptide comprising the sequence set forth in SEQ ID NO: 66;
said β subunit polypeptide comprising the sequence set forth in SEQ ID NO: 53;
said α subunit polypeptide and β subunit polypeptide are linked by a peptide bond;
said β subunit polypeptide comprises a seatbelt region that wraps around said alpha α subunit polypeptide;
said α and β subunit polypeptides are covalently linked via two disulfide bonds consisting of a first disulfide bond and a second disulfide bond;
said first disulfide bond is between cys29 of said α subunit polypeptide and a cysteine residue on the N-terminus end of said β subunit polypeptide; and
said second disulfide bond is between cys110 of said α subunit polypeptide and cys98 of said β subunit polypeptide.
2. The analog of claim 1 , wherein said α subunit polypeptide has reduced glycosylation relative to a native α subunit polypeptide.
3. The analog of claim 2 , wherein said α subunit polypeptide comprises an α2 loop which has reduced glycosylation relative to an α2 loop of a native α subunit polypeptide.
4. The analog of claim 3 wherein said α subunit polypeptide comprises a mutation of at least one asparagine residue relative to a native α subunit polypeptide.
5. The analog of claim 1 , wherein said α subunit polypeptide and β subunit polypeptide are linked by a peptide bond, wherein said peptide bond is between the C-terminus of said α subunit polypeptide and the N-terminus of said β subunit polypeptide.
6. The analog of claim 5 , wherein said analog comprises a cleavage site in between said α subunit and said β subunit.
7. The analog of claim 6 , wherein said cleavage site is selected from the group consisting of a furin cleavage site, thrombin cleavage site, Factor Xa cleavage site, and enterokinase cleavage site.
8. A nucleic acid comprising a polynucleotide encoding an α subunit polypeptide, wherein said α subunit polypeptide has an amino acid sequence comprising the sequence recited in SEQ ID NO: 66.
9. A nucleic acid comprising a polynucleotide encoding a β subunit polypeptide wherein said β subunit polypeptide has an amino acid sequence comprising the sequence recited in SEQ ID NO: 53.
10. A vector comprising a nucleic acid of claim 8 .
11. A vector comprising a nucleic acid of claim 9 .
12. An isolated host cell comprising, a nucleic acid of claim 8 .
13. An isolated host cell comprising a nucleic acid of claim 9 .
14. A method of inducing follicle development in fish comprising administering an effective dose of a formulation comprising an analog of claim 1 to said fish.