Variants of RANKL protein
The present invention relates to novel, soluble, recombinant variants RANKL (Receptor Activator of Nuclear Factor—κB Ligand) proteins, which may be expressed solubly in E. coli, variants that act as RANKL antagonists, and methods for generating the same.
1. A variant RANKL protein comprising amino acid substitution C221S and at least one substitution selected from the group consisting of: A232K, I207K, I207R, I247A, I247D, I247E, I247K, I247Q, and I247R in SEQ ID NO:1 when numbered as amino acids 68-317 corresponding to the numbering of the full-length wild-type human RANKL sequence.
2. A variant RANKL protein comprising amino acid substitution C221S and at least one amino acid substitution selected from the group consisting of: I207K, I247A, I247D, I247E, I247K and I247Q in SEQ ID NO:1 when numbered as amino acids 68-317 corresponding to the numbering of the full-length wild-type human RANKL sequence.
3. A variant RANKL protein comprising amino acid substitutions C221S/I247E, and at least one set of substitutions selected from the group consisting of: A172R, A172Q, Y188E, Y188F, Y188M, T233D, T233E, S228D, I249Q, I249R, I249R/S228H, H253T, D302Q, S268D, R191K, S183K, H189R, Y188Q, H198E, K181Q, Q237K, K248E,R284E, I249R/S228E, D300N, D190T/K248E, R191Q, T227K, D190Q/K248E, E226R/i249R, R191E/1249R, H225E, H225N, H225T, R223Q, E226D, E226Q, E226R, E269Q, E269T, Q237E, K248E/T227Q, H225R/D1 90Q, H225R/G192A, H225R, H225N/G192A, E269K, F270T, H225N/I249R, H225R/I249R, and G192A/K298E in SEQ ID NO:1 when numbered as amino acids 68-317 corresponding to the numbering of the full-length wild-type human RANKL sequence.
4. A variant RANKL protein according to claims 1 , 2 or 3 , wherein said variant protein is chemically modified.
5. A RANKL variant protein according to claim 4 , wherein said chemical modification is PEGylation.
6. A RANKL variant protein according to claim 4 , wherein said chemical modification is glycosylation.
7. A recombinant nucleic acid encoding the protein of claim 1 , 2 or 3 .
8. An isolated host cell comprising the recombinant nucleic acid of claim 7 .
9. A method of producing a non-naturally occurring RANKL protein comprising culturing the host cell of claim 8 under conditions suitable for expression of said nucleic acid.
10. A method according to claim 9 , further comprising recovering said RANKL protein.
11. A composition comprising a variant RANKL protein according to claims 2 or 3 and a carrier.
12. A method of making a mixed RANKL oligomer comprising contacting at least one variant RANKL protein comprising at least a variant extracellular domain of a RANKL monomer protein with a homo-oligomer comprising naturally occurring RANKL monomer proteins, under conditions whereby at least one naturally occurring RANKL monomer exchanges with a variant monomer to form a mixed oligomer, and wherein the variant RANKL protein comprises the variant RANKL protein of claim 1 , 2 or 3 .