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
1999-04-28
2002-09-17
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254200, C435S254210, C435S254230
Reexamination Certificate
active
06451557
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production of hydroxylated triple helical proteins such as natural and synthetic collagens, natural and synthetic collagen fragments, and natural and synthetic collagen-like proteins, by recombinant DNA technology. In particular, the invention relates to a method for producing hydroxylated triple helical proteins in yeast host cells by introducing to a suitable yeast host cell, DNA sequences encoding the triple helical protein as well as prolyl 4-hydroxylase (P4H), in a manner wherein the introduced DNA sequences are stably retained and segregated by the yeast host cells.
BACKGROUND OF THE INVENTION
The collagen family of proteins represents the most abundant protein in mammals, forming the major fibrous component of, for example, skin, bone, tendon, cartilage and blood vessels. Each collagen protein consists of three polypeptide chains (alpha chains) characterised by a (Gly-X-Y)
n
repeating sequence, which are folded into a triple helical protein conformation. Type I collagen (typically found in skin, tendon, bone and cornea) consists of two types of polypeptide chain termed &agr;1(I) and &agr;2(I) [i.e. &agr;1(I)
2
&agr;2(I)], while other collagen types such as Type II [&agr;1(ll)
3
] and Type III [&agr;1(III)
3
] have three identical polypeptide chains. These collagen proteins spontaneously aggregate to form fibrils which are incorporated into the extracellular matrix where, in mature tissue, they have a structural role and, in developing tissue, they have a directive role. The collagen fibrils, after cross-linking, are highly insoluble and have great tensile strength.
The ability of collagen to form insoluble fibrils makes them attractive for numerous medical applications including bioimplant production, soft tissue augmentation and wound/burn dressings. To date, most collagens approved for these applications have been sourced from animal sources, primarily bovine. While such animal-sourced collagens have been successful, there is some concern that their use risks serious immunogenicity problems and transmission of infective diseases and spongiform encephalopathies (e.g. bovine spongiform encephalopathy (BSE)). Accordingly, there is significant interest in the development of methods of production of collagens or collagen fragments by recombinant DNA technology. Further, the use of recombinant DNA technology is desirable in that it allows for the potential production of synthetic collagens and collagen fragments which may include, for example, exogenous biologically active domains (i.e. to provide additional protein function) and other useful characteristics (e.g. improved biocompatability and stability).
The in vivo biosynthesis of collagen proteins is a complex process involving many post translational events. A key event is the hydroxylation by the enzyme prolyl 4-hydroxylase (P4H) of prolyl residues in the Y-position of the repeating (Gly-X-Y)
n
sequences to 4-hydroxyproline. This hydroxylation has been found to be beneficial for nucleation of folding of triple helical proteins. For collagens, it is essential for stability at body temperature. Accordingly, the development of a commercially viable method for the production of recombinant collagen requires co-expression of P4H with the alpha chains. For mammalian host cells, co-expression of P4H will occur autonomously since these cells should naturally express P4H. However, for yeast host cells, which for reasons of cost, ease and efficiency are more attractive for expression of recombinant eukaryotic proteins, transformation with DNA sequences encoding P4H will also be required. Since P4H consists of &agr; and &bgr; subunits of about 60 kDa and 60 kDa, yeast host cells for expression of recombinant collagen will require co-transformation with at least three exogenous DNA sequences (i.e., encoding an alpha chain, P4H &agr; subunit and P4H &agr; subunit) and stability problems would therefore be expected if cloned on three separate vectors or, alternatively, all on episomal type vector. Indeed, even under continuous selection pressure, many episomal type vectors suffer stability problems if they are large or are present at relatively low copy number. An object of the present invention is therefore to provide a method for expressing recombinant collagen and other triple helical proteins from yeast host cells wherein the introduced DNA sequences are stably retained and segregated independent of continuous selection pressure.
SUMMARY OF THE INVENTION
Thus, in a first aspect, the present invention provides a method of producing a hydroxylated triple helical protein in yeast comprising the steps of:
introducing to a suitable yeast host cell a first nucleotide sequence encoding P4H &agr; subunit, a second nucleotide sequence encoding P4H &bgr; subunit and one or more product-encoding nucleotide sequences which encode(s) a polypeptide(s) or peptide(s) which, when hydroxylated, form the said hydroxylated triple helical protein, each of said first, second and product-encoding nucleotide sequences being operably linked to promoter sequences, and
culturing said yeast host cell under conditions suitable to achieve expression of said first, second and product-encoding nucleotide sequences to thereby produce said hydroxylated triple helical protein; wherein said method is characterised in that the step of introducing the first, second and product-encoding nucleotide sequences results in the said first, second and product-encoding nucleotide sequences, together with their respective operably linked promoter sequences, being borne on one or more replicable DNA molecules that are stably retained and segregated by said yeast host cell during said step of culturing.
In a second aspect, the present invention provides a yeast host cell capable of producing a hydroxylated triple helical protein, said yeast host cell including a first nucleotide sequence encoding P4H &agr; subunit, a second nucleotide sequence encoding P4H &bgr; subunit and one or more product-encoding nucleotide sequences which encode(s) a polypeptide(s) or peptide(s) which, when hydroxylated, form the said hydroxylated triple helical protein, each of said first, second and product-encoding nucleotide sequences being operably linked to promoter sequences, and wherein said first, second and product-encoding nucleotide sequences, together with their respective operably linked promoter sequences, are borne on one or more replicable DNA molecules that are stably retained and segregated by said yeast host cell.
In a third aspect, the present invention provides a triple helical protein produced in accordance with the method of the first aspect.
In a fourth aspect, the present invention provides a biomaterial or therapeutic product comprising a triple helical protein produced in accordance with the method of the first aspect.
DETAILED DISCLOSURE OF THE INVENTION
The method according to the invention requires that the first and second nucleotide sequences encoding the P4H &agr; and &bgr; subunits and the product-encoding nucleotide sequences be introduced to a suitable yeast host cell in a manner such that they are borne on one or more DNA molecules that are stably retained and segregated by the yeast host cell during culturing. In this way, all daughter cells will include the first, second and product-encoding nucleotide sequences and thus stable and efficient expression of a hydroxylated triple helical protein product can be ensured throughout the culturing step and without the use of continuous selection pressure.
The method according to the invention can be achieved by; (i) integrating (e.g. by homologous recombination) one or more of the exogenous nucleotide sequences (i.e. one or more of the first, second and product-encoding nucleotide sequences) into one or more chromosome(s) of the yeast host cell, or (ii) including one or more of the exogenous nucleotide sequences within one or more vector(s) including a centromere (CEN) sequence(s). Alternatively, a combination of these techniques may be u
Galanis Maria
Ramshaw John Alan Maurice
Vaughan Paul Richard
Werkmeister Jerome Anthony
Commonwealth Scientific and Industrial Research Organisation
Schwartzman Robert A.
Sughrue & Mion, PLLC
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