Recombinant bone morphogenetic protein heterodimers, compositions and methods of use
First Claim
1. A method for producing a heterodimeric protein having bone stimulating activity comprising culturing under suitable conditions to produce said protein a selected host cell containing a first DNA sequence encoding a BMP selected from the group consisting of BMP-2 and BMP-4 and a second DNA sequence encoding BMP-5, said sequences each being under the control of a suitable regulatory sequence capable of directing co-expression of said proteins, and isolating said heterodimeric protein from the culture medium.
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Abstract
The present invention relates to a methods for producing recombinant heterodimeric BMP proteins useful in the field of treating bone defects, healing bone injury and in wound healing in general. The invention also relates to the recombinant heterodimers and compositions containing them.
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Citations
24 Claims
- 1. A method for producing a heterodimeric protein having bone stimulating activity comprising culturing under suitable conditions to produce said protein a selected host cell containing a first DNA sequence encoding a BMP selected from the group consisting of BMP-2 and BMP-4 and a second DNA sequence encoding BMP-5, said sequences each being under the control of a suitable regulatory sequence capable of directing co-expression of said proteins, and isolating said heterodimeric protein from the culture medium.
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10. A method for producing a heterodimeric protein having bone stimulating activity in a bacterial cell comprising culturing a selected host cell containing a first DNA sequence encoding a BMP selected from the group consisting of BMP-2 and BMP-4 under the control of a suitable regulatory sequence capable of directing expression of the protein under conditions suitable for the formation of a soluble, monomeric protein;
- isolating said monomeric protein from inclusion bodies and/or said culture medium;
culturing a selected host cell containing a second DNA sequence encoding BMP-5 under the control of a suitable regulatory sequence capable of directing expression of the protein or protein fragment under said conditions to form a second soluble, monomeric protein;
isolating said monomeric protein from inclusion bodies and/or said culture medium; and
mixing said soluble monomeric proteins under conditions permitting the formation of dimeric proteins associated by at least one covalent disulfide bond;
isolating from the mixture a heterodimeric protein. - View Dependent Claims (11, 12)
- isolating said monomeric protein from inclusion bodies and/or said culture medium;
- 13. An isolated recombinant heterodimeric protein having bone stimulating activity comprising a first BMP protein selected from the group consisting of BMP-2 and BMP-4 associated by at least one disulfide bond with a BMP-5 protein.
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16. An isolated protein comprising a recombinant heterodimeric protein having bone stimulating activity produced by co-expression in a selected host cell and isolation from the culture medium said protein comprising a subunit of a first BMP selected from the group consisting of BMP-2 and BMP-4, associated by at least one disulfide bond with a second subunit of a BMP-5.
- 17. A cell line comprising a nucleotide sequence encoding a first BMP selected from the group consisting of BMP-2 and BMP-4 under control of a suitable expression regulatory system and a nucleotide sequence encoding a BMP-5 under control of a suitable expression regulatory system, said regulatory systems capable of directing the co-expression of said BMPs and the formation of heterodimeric protein.
- 22. A DNA molecule comprising a sequence encoding a first BMP selected from the group consisting of BMP-2 and BMP4 and a sequence encoding a BMP-5 said sequences under the control of at least one suitable regulatory sequence capable of directing co-expression of each BMP.
Specification