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
2000-05-31
2002-07-02
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S024300, C530S350000, C530S354000, C530S356000
Reexamination Certificate
active
06413742
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to the field of recombinant protein production, and particularly to the production of telopeptide collagen in recombinant host cells.
BACKGROUND ART
Collagen is the major protein component of bone, cartilage, skin and connective tissue in animals. Collagen in its native form is typically a rigid, rod-shaped molecule approximately 300 nm long and 1.5 nm in diameter. It is composed of three collagen polypeptide monomers which form a triple helix. Mature collagen monomers are characterized by a long midsection having the repeating sequence-Gly-X-Y, where X and Y are often proline or hydroxyproline, bounded at each end by the “telopeptide” regions, which constitute less than about 5% of the molecule. The telopeptide regions of the chains are typically responsible for the crosslinking between the chains (i.e., the formation of collagen fibrils), and for the immunogenicity of the protein. Collagen occurs naturally in a number of “types”, each having different physical properties. The most abundant types in mammals and birds are types I, II and III.
Mature collagen is formed by the association of three procollagen monomers which include “pro” domains at the amino and carboxy terminal ends of the polypeptides. The pro domains are cleaved from the assembled procollagen trimer to create mature, or “telopeptide” collagen. The telopeptide domains may be removed by chemical or enzymatic means to create “atelopeptide” collagen.
Interestingly, although there are a large number of different genes encoding for different procollagen monomers, only particular combinations are produced naturally. For example, skin fibroblasts synthesize 10 different procollagen monomers (pro&agr;1(I), pro&agr;1(III), pro&agr;1(V), pro&agr;2(I), pro&agr;2(V), pro&agr;3(V), pro&agr;1(VI), pro&agr;2(VI), pro&agr;3(VI) and pro&agr;1(VII)), but only 5 types of mature collagen are produced (types I, III, V, VI and VII).
Collagen has been utilized extensively in biological research as a substrate for in vitro cell culture. It has also been widely used as a component of biocompatible materials for use in prosthetic implants, sustained drug release matrices, artificial skin, and wound dressing and wound healing matrices.
Historically, collagen has been isolated from natural sources, such as bovine hide, cartilage or bones, and rat tails. Bones are usually dried, defatted, crushed, and demineralized to extract collagen, while cartilage and hide are typically minced and digested with proteolytic enzymes other than collagenase. As collagen is resistant to most proteolytic enzymes (except collagenase), this procedure can conveniently remove most of the contaminating protein that would otherwise be extracted along with the collagen. However, for medical use, species-matched collagen (e.g., human collagen for use in human subjects) is highly desirable in order to minimize the potential for immune response to the collagen material.
Human collagen may be purified from human sources such human placenta (see, for example, U.S. Pat. Nos. 5,002,071 and 5,428,022). Of course, the source material for human collagen is limited in supply and carries with it the risk of contamination by pathogens such as hepatitis virus and human immunodeficiency virus (HIV). Additionally, the material recovered from placenta is biased as to type and not entirely homogenous.
Collagen may also be produced by recombinant methods. For example, International Patent Application No. WO 97/14431 discloses methods for recombinant production of procollagen in yeast cells and U.S. Pat. No. 5,593,859 discloses the expression of procollagen genes in a variety of cell types. In general, the recombinant production of collagen requires a cloned DNA sequence encoding the appropriate procollagen monomer(s). The procollagen gene(s) is cloned into a vector containing the appropriate DNA sequences and signals for expression of the gene and the construct is introduced into the host cells. Optionally, genes expressing a prolyl-4-hydroxylase alpha subunit and a protein disulfide isomerase are also introduced into the host cells (these are the two subunits which make up prolyl4-hydroxylase). Addition of the prolyl-4-hydroxylase leads to the conversion of some of the prolyl residues in the procollagen chains to hydroxyproline, which stabilize the triple helix and increase the thermal stability of the protein.
Alternately, recombinant collagen may be produced using transgenic technology. Constructs containing the desired collagen gene linked to the appropriate promoter/enhancer elements and processing signals are introduced into embryo cells by the formation of ES cell chimera, direct injection into oocytes, or any other appropriate technique. Transgenic production of recombinant collagen is particularly advantageous when the collagen is expressed in milk (i.e., by mammary cells), such as described in U.S. Pat. No. 5,667,839 to Berg. However, the production of transgenic animals for commercial production of collagen is a long and expensive process.
One difficulty of recombinant expression of collagen is the processing of the “pro” regions of procollagen monomers. It is widely accepted that folding of the three monomers to form the trimer begins in the carboxyl pro-region (“C propeptide”) and that the C propeptide contains signals responsible for monomer selection (Bachinger et al., 1980,
Eur. J. Biochem.,
106:619-632; Bachinger et al., 1981,
J. Biol. Chem.
256:13193-13199). One group has identified a region in the carboxy pro-region that they believe is necessary and sufficient for monomer selection (Bulleid et al., 1997,
EMBO J.
16(22):6694-6701; Lees et al., 1997,
EMBO J.
16(5):908-916; International Patent Application No. WO 97/08311; McLaughling et al., 1998,
Matrix Biol.
16:369-377). Additionally, Lee et al. (1992,
J. Biol. Chem.
267(33):24126-24133) have shown that deletion of the N propeptide results in decreased secretion of human &agr;1 pC collagen from CHL cells, but not Mov-13 cells. Accordingly, it is believed that the pro-regions must be retained for proper chain selection, alignment and folding of collagen produced by recombinant methods. In cells which normally produce collagens, specific proteolytic processing enzymes are produced which remove the N and C propeptides following the secretion of collagen. These enzymes are not present in cells which do not normally produce procollagen (including commonly used recombinant host cells such as bacteria and yeast).
Ideally, the recombinant production of collagen is accomplished with a recombinant host cell system that has a high capacity and a relatively low cost (such as bacteria or yeast). Because bacteria and yeast do not normally produce the enzyme necessary for processing of the N and C propeptides, the propeptides must be removed after recovering the recombinant procollagen from the host cells. This can be accomplished by the use of pepsin or other proteolytic enzymes such as PRONASE® or trypsin, but in vitro processing produces “ragged” ends that do not correspond to the ends of mature collagen secreted by mammalian cells which normally produce fibrillar collagen. Alternately, the enzymes which process the N and C propeptides can be produced and used to remove the propeptides. Any contamination of these enzyme preparations with other proteases will result in ragged ends. This added processing step increases the cost and decreases the convenience of production in these otherwise desirable host cell systems.
Gelatin can be considered a collagen derivative. Gelatin is denatured collagen, generally in monomeric form, which may be fragmented as well. Gelatin serves a large number of uses, particularly in foodstuffs as well as in medicine, where it is frequently used for coating tablets or for making capsules. However, the possibility of the spread of prion-based diseases through animal-derived gelatin has made the use of animal-derived gelatin less attractive.
Accordingly, there is a need in the art for simplified methods of producing gelatin and genuine telop
Chang Robert
Chisholm George
Hitzeman Ronald A.
McMullin Hugh
Olsen David R.
Cohesion Technologies, Inc.
Osman Richard Aron
Whisenant Ethan C.
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