Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-07
2001-08-21
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S455000, C530S300000, C530S350000, C530S356000
Reexamination Certificate
active
06277600
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel chimera protein comprising a collagen having covalently bonded thereto a biologically active peptide such as a cell growth factor or a cytokine; and a method for producing such chimera protein. This invention is also directed to a novel collagen matrix produced through fibril-formation of such chimera protein.
BACKGROUND ART
Collagen is a protein which is widely used in medical field as a biomaterial for restoring the body damage and as a carrier of various drugs. The collagens currently used for such applications are collagens of animal origin extracted from the tissue of cows, pigs, and the like. Use of a collagen of human origin is desirable in consideration of immunological reactions, and also, contamination of pathogens such as viruses and prions from animal tissues which is currently a serious issue. In view of such situation, the inventors of the present invention have proposed a method for producing a recombinant human collagen having a triple helical structure equivalent to the one found in human body by infecting insect cells with a recombinant virus having the cDNA coding for a human collagen inserted therein (JP-A 8-23979). This method is also described in Tomita et al., (Biochemical J. 312, 847-853 (1995) and J. Biochem. 121, 1061-1069 (1997)). And the similar methods are described in Lamberg et al., (J. Biol. Chem. 271, 11988-11995 (1996)), Myllyharju et al., (J. Biol. Chem. 272, 21824-21830 (1997)), Nokelainen et al., (Matrix Biology, 16, 329-338 (1998)).
Prockop et al. have also proposed a method for producing recombinant human collagens by using mammal cells or yeasts (JP-A 7-501939, Published Japanese Translation of WO93/07889). Ala-Kokko et al., (J. Biol. Chem. 266, 14175-14178 (1991)), Geddis et al., (Matrix 13, 399-405 (1993)) and Fertala et al., (Biochemical, J. 298, 31-37 (1994)) disclose the similar methods.
It is not clear whether a plurality of proteins having formed as fusion protein can always exhibit their own activities, because the activity of a protein is dependent on its conformation. Further, it is not clear whether a trimer of the fusion protein can be formed which retains the activities of the collagen and the peptide, because trimeric fusion protein containing essentially full length of collagen has not been heretofore reported.
On the other hand, cell growth factors, cytokines and various other biologically active peptides are highly hoped for use as a drug. These peptides, however, suffer from high elimination rate, and practical use of such biologically active peptides is often prevented by their high elimination rate. In order to obviate such situation, mixing of the biologically active peptide with the collagen and embedding of the mixture in the body for controlled release has been proposed (Fujioka et al., J. Controlled Release 33, 307 to 315, 1995). This reference describes that when interferon (INF) was mixed with gelatin or collagen and embedded in animal body the INF release from gelatin gel was 100% after 1 day, but the release from collagen gel was less than 100% though more than 60% after 1 day. It also describes that drying of the mixture of INF and collagen reduces the release rate of INF.
DISCLOSURE OF THE INVENTION
As described above, collagen is expected for its use as a carrier of various biologically active peptides which enable controlled release of the peptides. Affinity of the collagen for the biologically active peptide is not necessarily high, and the biologically active peptide is not stably retained in the collagen matrix for a prolonged period of time by merely mixing the biologically active peptides with the collagen. When the biologically active peptide is chemically bonded to the collagen using glutaraldehyde or the like, the biologically active peptide will undergo total or partial inactivation.
To solve the problem the present inventors have been studied whether the peptide of collagen and biologically active peptide could be produced as a single protein or not.
In view of the situation as described above, an object of the present invention is to provide a method for fusing a biologically active peptide to a collagen with no loss in the activity, and hence, to provide a novel trimeric chimera protein which is simultaneously provided with the features of the biologically active peptide and the collagen. Another object of the invention is to provide a novel collagen matrix produced by polymerizing the chimera protein.
In order to obviate the situation as described above, the inventors of the present invention have invented a method for producing a recombinant chimera protein wherein the chimera protein is produced by ligating the gene coding for a procollagen and the gene coding for a biologically active peptide by genetic engineering means, and the ligation product is expressed by using a recombinant protein expression system. As described above, various methods are reported to produce collagen with native conformation by recombinant method. In these reports there are not any indications of the technical possibilities to produce a chimera protein of biologically active peptide and collagen. There have been no attempt to produce such a chimera protein in the prior art.
Furthermore, the inventors invented a collagen matrix comprising collagen fiber which is produced through polymerizing such chimera protein either alone or after mixing with collagen molecule. The present invention was thereby completed. In other words, the objects of the present invention are attained by means of (1) to (14) as described below.
(1) A trimeric chimera protein comprising a collagen and a biologically active peptide fused to the collagen, wherein said biologically active peptide is fused on the side of the amino terminal of the collagen, and the collagen is a fibril-forming collagen having a triple helical structure in its triple helix region.
In this invention, biologically active peptide means a peptide having a biological activity to vary all kinds of cellular function, such as proliferation, migration, gene expression, differentiation and the like.
(2) A trimeric chimera protein according to the above (1) wherein said trimeric chimera protein substantially lacks carboxyl propeptide region of the procollagen which is a collagen precursor; or said carboxyl propeptide region has been substituted with a peptide which does not inhibit fibril-formation and which is capable of forming the urimer.
(3) A trimeric chimera protein according to the above (1) or (2) wherein said biologically active peptide is directly fused to the amino terminal of the triple helix region of the collagen; or a part of the amino propeptide region of the collagen precursor procollagen is inserted at the fusion site of the biologically active peptide and the collagen.
(4) A trimeric chimera protein according to the above (1) or (2) wherein full or substantially full length of the amino propeptide region of the procollagen is inserted in the fusion site of the biologically active peptide and the collagen.
(5) A collagen matrix comprising collagen fiber containing a chimera protein comprising a collagen and a biologically active peptide fused to the amino terminal of the collagen as its constituent.
(6) A collagen matrix according to the above (5) wherein said collagen fiber comprises a mixture of a chimera protein and a collagen molecule.
(7) A collagen matrix according to the above (6) wherein said chimera protein is uniformly distributed in the collagen fiber, and the portion of said biologically active peptide is embedded in the collagen fiber.
(8) A collagen matrix according to the above (7) wherein said chimera protein is a trimeric chimera protein of any one of the above (1) to (3).
(9) A collagen matrix according to the above (6) wherein said chimera protein is distributed in the surface region of said collagen fiber, and said biologically active peptide protrudes beyond the surface of said collagen fiber.
(10) A collagen matrix according to the above (9) wherein said chimera protein is a trimeric chim
Kitajima Takashi
Tomita Masahiro
Burns Doane , Swecker, Mathis LLP
Horlick Kenneth R.
Terumo Kabushiki Kaisha
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