US6190880B1 — Recombinant bone morphogenetic protein heterodime…

Claim 1

Independent Claim 76 words

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.

Claim 2

Independent Claim 76 words

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.

Claim 3

Dependent Claim 36 words

The method according to claim 1 wherein said first DNA is present on a first vector transfected into said host cell and said second DNA is present on a second vector transfected into said host cell.

Claim 4

Dependent Claim 19 words

The method according to claim 1 wherein both said BMPs are integrated into a chromosome of said host cell.

Claim 5

Dependent Claim 15 words

The method according to claim 1 wherein both BMPs are present on a single vector.

Claim 6

Dependent Claim 24 words

The method according to claim 2 wherein more than a single copy of the gene encoding each said BMP is present on each vector.

Claim 7

Dependent Claim 64 words

The method according to claim 1 wherein said host cell is a hybrid cell prepared by culturing two fused selected, stable host cells, each host cell transfected with a DNA sequence encoding said selected first or second BMP, said sequences under the control of a suitable regulatory sequence capable of directing expression of each protein, and isolating said heterodimer protein from the culture medium.

Claim 8

Dependent Claim 14 words

The method according to claim 1 wherein said host cell is a mammalian cell.

Claim 9

Dependent Claim 14 words

The method according to claim 1 wherein said host cell is an insect cell.

Claim 10

Dependent Claim 14 words

The method according to claim 1 wherein said host cell is a yeast cell.

Claim 11

Independent Claim 155 words

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.

Claim 12

Independent Claim 155 words

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.

Claim 13

Dependent Claim 13 words

The method according to claim 10 wherein said host cell is E. coli.

Claim 14

Dependent Claim 17 words

The method according to claim 10 wherein said conditions comprise treating said protein with a solubilizing agent.

Claim 15

Dependent Claim 34 words

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.

Claim 16

Dependent Claim 34 words

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.

Claim 17

Dependent Claim 12 words

The protein according to claim 13 wherein said first protein is BMP-2.

Claim 18

Dependent Claim 12 words

The protein according to claim 13 wherein said first protein is BMP-4.

Claim 19

Dependent Claim 58 words

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.

Claim 20

Dependent Claim 58 words

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.

Claim 21

Dependent Claim 60 words

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.

Claim 22

Dependent Claim 60 words

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.

Claim 23

Dependent Claim 21 words

The cell line according to claim 17 wherein said nucleotide sequences encoding BMP proteins are present in a single DNA molecule.

Claim 24

Dependent Claim 37 words

The cell line according to claim 17 wherein said nucleotide sequence encoding said first BMP is present on a first DNA molecule and said nucleotide sequence encoding said second BMP is present on a second DNA molecule.

Claim 25

Dependent Claim 38 words

The cell line according to claim 18 wherein said single DNA molecule comprises a first transcription unit containing a nucleic acid encoding said first BMP and a second transcription unit containing a nucleic acid encoding said second BMP.

Claim 26

Dependent Claim 39 words

The cell line according to claim 18 wherein said single DNA molecule comprises a single transcription unit containing multiple copies of said nucleic acid encoding said first BMP and multiple copies of said nucleic acid encoding said second BMP.

Claim 27

Dependent Claim 44 words

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.

Claim 28

Dependent Claim 44 words

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.

Claim 29

Dependent Claim 32 words

The molecule according to claim 22 comprising a first transcription unit containing a nucleic acid encoding said first BMP and said second transcription unit containing a nucleic acid encoding said second BMP.

Claim 30

Dependent Claim 33 words

The molecule according to claim 22 comprising a single transcription unit containing multiple copies of said nucleic acid encoding said first BMP and multiple copies of said nucleic acid encoding said second BMP.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2004-02-20	2004-08-21	2005-02-20	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2008-02-20	2008-08-21	2009-02-20	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2012-02-20	2012-08-21	2013-02-20	$7700.00	Window Closed / Lapsed

Recombinant bone morphogenetic protein heterodimers, compositions and methods of use

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