Method of enhancing recombinant protein production
The present invention provides a method of enhanced protein production that comprises the step of expressing a recombinant gene encoding the protein in eukaryotic cells under conditions in which cleavage of the pro-domain of the protein is inhibited or eliminated. Generally the method of the present invention includes the step of inhibiting or altering the cleavage of a pro-domain of a recombinant protein of interest in order to increase the amount of recombinant protein secreted from a eukaryotic cell. Recombinant proteins that can be prepared using the method of this invention include members of the transforming growth factor-β (TGF-β) superfamily, such as bone morphogenetic proteins. Also provided are genetically engineered cells and polynucleotides for performing the method of the invention.
1. A method of increasing the production yield of a recombinant protein having a pro-domain, wherein the recombinant protein is bone morphogenetic protein 2 (BMP-2), the method comprising the step of expressing a nucleotide sequence comprising a recombinant gene encoding said recombinant protein in eukaryotic cells and wherein cleavage of the pro-domain of the protein is inhibited or eliminated by mutating the recombinant gene to make the protein resistant to cleavage by a proprotein convertase, wherein the proprotein convertase recognition site REKR, consisting of amino acid residues 279 to 282 of SEQ ID NO: 2, of the recombinant protein is replaced with the amino acid sequence of SEQ ID NO: 16, and wherein said increased production yield is in comparison to the yield obtained using a method in which the cleavage of said pro-domain is not inhibited or eliminated.
2. The method according to claim 1 , wherein the bone morphogenetic 2 protein is a mammalian BMP-2.
3. The method according to claim 2 , wherein the mammalian BMP-2 is human BMP-2 (hBMP-2).
4. The method according to claim 1 , wherein where the eukaryotic cells are mammalian cells.
5. The method according to claim 4 , wherein the mammalian cells are derived from cell lines.
6. The method according to claim 5 , wherein the cell lines are CHO, HEK293, or COS cell lines.
7. The method according to claim 1 , wherein the proprotein convertase is a mammalian proprotein convertase and wherein the mammalian proprotein convertase is Furin, PC1, PC2, PACE 4, PC4, PC5 or PC7.
8. The method according to claim 1 , wherein the proprotein convertase is Furin.