Method for producing, in yeast, a hydroxylated triple helical protein, and yeast host cells useful in said method

Abstract

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 (PH4), in a manner wherein the introduced DNA sequences are replicated, stably retained and segregated by the yeast host cells.

Description

[0001]This is a Continuation of application Ser. No. 10 / 023,831, filed Dec. 21, 2001; which is a Continuation-in-part Application of application Ser. No. 09 / 297,269, filed Apr. 28, 1999 (now U.S. Pat. No. 6,451,557); which in turn is a National stage filing under 35U.S.C. § 371 of PCT / AU97 / 00721, filed Oct. 29, 1997; the entire disclosure of each of which is incorporated herein by reference.FIELD OF THE INVENTION[0002]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 replicated, stably ret...

AI Technical Summary

Benefits of technology

[0044]The method according to the invention requires that the first and second nucleotide sequences encoding the P4H α and β subunits and the third nucleotide sequence (i.e. the “product-encoding nucleotide sequence”) be introduced to a suitable yeast host cell in a manner such that they are borne on one or more DNA molecules that are replicated, 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.

[0052]Alternatively, the portion of the product-encoding nucleotide sequence(s) encoding the repeat unit of domain Z may be generated using DNA ligase to join non-palindromic nucleotide sequences, which may be produced by PCR techniques or chemical DNA synthesis, end to end in such a manner as to maintain the open reading frame of the product-encoding nucleotide sequence and which ensures that every third amino acid of the encoded Z domain is Gly. The use of complimentary, but non-palindromic, overhanging sequences at the ends of the designed non-palindromic nucleotide sequences ensures that they are joined in a consistent head to tail orientation. Further, the strategy allows the ready linking of nucleotide sequences encoding other polypeptide or peptide domains (e.g. the abovementioned A, B, C and / or D), by utilising terminal non-palindromic overhang sequences which result in the generation of a restriction enzyme site. This restriction enzyme site can then be used for the additional cloning of nucleotide sequences encoding the other polypeptide or peptide domains.

[0055]The product-encoding nucleotide sequence(s) also comprise a nucleotide sequence preferably selected to match the codon use preferences of the selected yeast expression host, and is also constructed so as to minimise the potential for GGG to CCC interactions that may destabilize the structure.

[0067]The method of the present invention enables the production of triple helical protein products with two or more repeat units, which allows control of biological function and permits the possibility of enhancing the efficacy of selected domains by, for example, increasing binding sites and avidity for interacting agents and by activation through receptor clustering.

Problems solved by technology

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)).

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.

Images