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

a technology of which is applied in the field of producing, in yeast, a hydroxylated triple helical protein and yeast host cells useful in said method, can solve the problems of many episomal type vectors suffering stability problems, their use risks serious immunogenicity problems, and the transmission of infective diseases and spongiform encephalopathies

Inactive Publication Date: 2008-06-12
COMMONWEALTH SCI & IND RES ORG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011]Z is a domain comprising two

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

Method used

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

Examples

Experimental program
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Effect test

example 1

Construction of a Yeast Vector for Co-Ordinated Co-Expression of the a and β Subunits of Prolyl-4-Hydroxylase

Production of Yeast Expression Vector:

[0078]pYEUra3 (Clontech) contains the bidirectional promoter for GAL 1-10 expression. Induction by galactose in the absence of glucose results in high level expression from pGAL1 of any protein encoded by DNA sequences inserted in the correct orientation in the MCS (multiple cloning site) [either XhoI, SalI, XbaI or BamHI sites] provided there is an initiating ATG start codon. For pGAL10, expression induced by galactose occurs if the DNA sequences to be expressed are inserted in frame with the ATG codon of GAL 10 when said DNA sequences to be expressed is inserted in the EcoRI site.

[0079]In order to utilise the EcoRI site for cloning, without the necessity that the insert be in frame with the ATG of GAL10 for expression, it was necessary to modify pYEUra3 to remove the GAL10 initiation codon. This was done as follows. A PCR fragment was g...

example 2

Co-ordinated Co-Expression of a Collagen Segment and Prolyl-4-Hydroxylase (α and β Subunit) and Synthesis of Hydroxylated Collagen TYPE III in Yeast

[0083]A 1.6 kbp recombinant collagen fragment was generated by PCR using primers 1989 [Forward primer 5′-gct.agc.aag.ctt GGA.GCT.CCA. GGC.CCA.CTT.GGG.ATT.GCT.GGG-3′ (SEQ ID NO: 15)] and 1903 [Reverse primer 5′-tcg.cga.tct.aga.TTA.TAA.AAA.GCA.AAC.AGG.GCC.AAC.GTC.CAC. ACC-3′ (SEQ ID NO:16)] homologous to a region of the collagen type III alpha 1 chain (COL3A1). The template for isolation of the fragment of type III collagen alpha 1 chain was prepared from Wizard purified DNA obtained from a cDNA library [HL1123n Lambda Max 1 Clontech Lot#1245, Human Kidney cDNA 5′-Stretch Library].

[0084]The actual size of the isolated 1.6 kbp fragment is 1635 bp, comprising 1611 bp of COL3A1 DNA flanked either side by 12 bp derived from the primers. The 1611 bp of COL3A1 DNA corresponds to nucleotides #2713-4826 (i.e. codon #905-1442) of the full-length co...

example 3

Use of Yeast Artificial Chromosomes [YACs] for Co-Ordinated expression of the α and β Subunits of Prolyl-4-Hydroxylase [P4H]

[0101]pYAC5 [11454 bp] (Kuhn and Ludwig, 1994) was digested with BamHI to liberate the HIS3 gene [1210 bp] from between the 2 telomere ends and with SalI-NruI to produce two fragments [left arm: fragment 1, 5448 bp & right arm: fragment2, 4238 bp] which were gel purified. Fragment 1 was BamHI-telomere end-E. coli ori-β-lactamase gene [ampicillin-resistance]-TRP1-ARS1-CEN4-tRNAsup-o-SalI. Fragment 2 was BamHI-telomere end-URA3-NruI.

[0102]pYEUra3.2.12β#39α#5 was digested with SalI-EcoRV to produce a P4H expression cassette fragment of the form SalI-XbaI-BamHI-α-ATG-BamHI-pGAL1-10-EcoRI-ATG-β-EcoRI-SmaI-EcoRV [4864 bp] which was gel purified. The expression cassette fragment encoding the cc and 13 subunits of P4H under the control of a galactose inducible bidirectional promoter was ligated with fragments 1 and 2 of the BamHI-SalI-NruI digested pYAC5 and the ligati...

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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...

Claims

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Application Information

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IPC IPC(8): A61K38/00C12P21/04C12N5/06C07K16/00C12N15/09A61K38/17A61K48/00C07K14/78C12N1/19C12N9/02C12N15/12C12P21/02C12R1/84
CPCA61K38/00C12P21/02C12N9/0071C07K14/78
Inventor VAUGHAN, PAUL RICHARDGALANIS, MARIARAMSHAW, JOHN ALAN MAURICEWERKMEISTER, JEROME ANTHONY
Owner COMMONWEALTH SCI & IND RES ORG
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