Expression of osteogenic factor OP-1 in cells of spodoptera frug

Details Expression of osteogenic factor OP-1 in cells of spodoptera frug Expression of osteogenic factor OP-1 in cells of spodoptera frug

1994-09-22

1997-06-24

Walsh, Stephen G.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 912, 4352351, 435348, 4353201, 935 32, C12P 1934, C12P 2106, C12N 500, C12N 1500

Patent

active

056416490

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to a process for obtaining human osteogenic protein, OP-1, via recombinant DNA technology. This process is exemplified by infecting cells of Spodoptera frugiperda with a recombinant baculovirus to produce the mature, biologically active form of OP-1.
2. Description of Related Art
A. OSTEOGENIC FACTOR OP-1
Osteogenic factor OP-1 belongs, in view of its biological activity, to the family of Bone Morphogenic Proteins (BMPs), which are factors responsible for the induction of cartilage and bone.
BMPs form part of the large superfamily of TGF-.beta. (Transforming Growth Factor .beta.), a family that includes embryonic morphogens, endocrine function regulators, wide-range regulators, and regulators that are specific for cell proliferation and differentiation.
TGF-.beta. is a prototype of this family. It is a dimer of two identical chains of 112 amino acids held together by disulfide bridges. Each chain is synthesized starting from a longer precursor of about 390 amino acids which has the characteristics of a secretory polypeptide, presenting a hydrophobic sequence in the N-terminal region which should function as a secretory peptide for the secretion of the molecule. The precursor is then processed to its mature form by cleavage by a specific peptidase, which cleaves four basic amino acids immediately prior to the biologically active domain. The precursor region plays an essential role in the correct folding of the mature portion in vivo, to the extent that to date, no mature, biologically active peptides are known to have been produced in Escherichia coli by recombinant DNA techniques.
These factors are known in various animal species from Drosophila to humans, their sequences having been maintained to a great extent throughout evolution. The sequence homology among the various polypeptides is very high, and resides mainly in the C-terminal region. The degree of identity of sequence varies between 25 and 90% among the various family members. In this region, between 7 and 9 cysteines are conserved among all the members. These are involved in the formation of disulfide bridges between the amino-acid chains.
BMPs induce chemotactic, proliferative and differential responses, which culminate in the transient formation of cartilage, followed by the accumulation of bone with hematopoietic marrow. The activity of BMPs has been characterized as strictly linked with the demineralized bone matrix, and is extractable with denaturing agents. They have in fact been extracted from various species including humans, monkeys, cattle, rats and mice (Sampath, T. K., Reddi, A. H. 1983, PNAS 80, 6591-6595; Urist, M. D. et al. 1979, PNAS 76, 1828-1832). Most studies were carried out on BMPs derived from bovine bone, an abundant and easily obtainable source.
In 1988 Wozney et al. (Wozney, J. M. et al., 1988, Science 242, 1528-1534) recovered a biologically active protein fraction of about 30 kD from bovine bone that could be detected by polyacrylamide gel electrophoresis under nonreducing conditions. Following reduction of the disulfide bridges by chemical methods, polypeptides of 30, 18 and 16 kD were obtained (Wang, E. A. et al., 1988, PNAS 85, 9484-9488). This protein fraction was digested with trypsin, and the peptides obtained were separated by HPLC and sequenced. This information was used in the synthesis of DNA probes which were used to identify the bovine genome sequences encoding the various factors. Using portions of these sequences as probes, the human sequences coding for the homologous factors were obtained.
Much is now known about these factors (Wozney, J. M. et al., 1990, J. Cell. Sci. Suppl. 13, 149-156; Wozney, J. M., 1989, Progress in Growth Factor Research, 1, 267-280). Some were obtained via recombinant DNA techniques. Some examples of patents on growth factors belonging to the abovesaid classes, obtained by recombinant DNA techniques, include EP 409472, WO 9011366, WO 8800205, EP 212474, WO 9105863, and U.S. Pat. No. 4,743,679.
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Luckow et al, "Trends in the Development of Baculovirus Expression Vectors", Bio/Technology 6: 47-55 (Jan. 1988).
Celeste et al, "Identification of transfroming growth factor .beta. family . . . " PNAS 87: 9843-9847 (Dec. 1990).
Madisen et al "Expression of the Human Immunodeficiency Virus gag Gene in Insect Cells", Virology 158: 248-250 (May 1987).

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