Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-09-08
1997-12-09
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351721, 4352521, 4352542, 4353201, 536 231, 536 241, 935 22, 935 23, C12P 2106, C12N 1563, C12N 120, C07H 2104
Patent
active
056959613
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel promoter DNA, particularly a novel expression system comprising DNA having a sequence containing transcriptional and translational signals that promote the over production of recombinant proteins both in bacterial hosts (eg., Escherichia coli) and yeasts (eg., Saccharomyces cerevisiae); and to a novel cloning method that allows the insertion of a heterologous gene into a vector or expression cassette directly at the authentic translational start point of a promoter, with no deleterious changes being made to either the native 5'-UTR of a vector promoter or to the codons of the inserted gene; allowing production of that promoter DNA. The design of the expression system lends itself to this unique strategy which allows heterologous genes to be directly cloned at a optimal position relative to the transcription/translation signals.
Particularly provided are expression cassettes comprising a sequence of the invention combined with a purpose built series of plasmids wherein the utility and efficiency of the resultant expression vectors can be demonstrated to over produce protein, particularly that of phenylalanine ammonia lyase (herein abbreviated to PAL), in E. coli and S. cerevisiae to levels hitherto unattainable.
BACKGROUND OF THE INVENTION
Although considerable progress has been made towards the development of expression systems for yeast (reviewed in Rose and Broach, 1990), the vectors lack the sophistication and versatility of their bacterial counterparts. Current vectors often contain many superfluous DNA sequences, which make them cumbersome and difficult to amplify and isolate in large quantities. The wealth of DNA present means that unique restriction sites are limited in number.
Yeast expression vectors are usually of the "sandwich" variety, whereby cloning sites are "sandwiched" between a homologous yeast promoter and transcriptional termination signals. The precise positioning of the cloning sites with respect to the authentic initiating codon (AUG) of the homologous yeast promoter represents something of a dilemma. If one chooses to place the cloning sites upstream to the AUG, then one inevitably disrupts the native 5'-untranslated region (5'-UTR) of the yeast promoter. Unavoidable insertion of heterologous untranslated sequence elements containing a high proportion of G residues, or elements creating secondary structures or containing the inserted AUG in a sub-optimal nucleotide context, can have catastrophic effects on expression levels, regardless of the strength of transcriptional activation signals (Donahue and Cigan, 1988; Baim and Sherman, 1988). For example, Bitter and Egan (1984) reported 10-15 fold lower expression levels of a Hepatitis B surface antigen (HBsAg) gene, fused to a yeast glyceraldehyde-3-phosphate (GPD) gene promoter, but utilising the native HBsAg 5' flanking region, compared to HBsAg fused to a GPD promoter and utilising the GPD 5' flanking region.
The alternative is to position the cloning sites immediately 3' to the authentic AUG of the yeast promoter. However, this has its own concomitant problems. Care must be taken that the fusion is "in frame", while the non-authentic amino terminus of the expressed protein may have unpredictable effects on its biological activity and antigenicity. These last two points render such fusion proteins unsuitable for use as a pharmaceutical without modification.
Preferably cloning is directly from the authentic AUG initiation codon. However, there has been no reported instance of a native yeast promoter with a usable restriction site encompassing its translational start point and the artificial creation of one would inevitably disrupt the start codon or its nucleotide context. The alternative is the lengthy and expensive procedure of chemically synthesizing an oligonucleotide "bridge" fragment that reaches from a convenient restriction site in the promoter 5' to the translational start to a site 3' to the ATG in the coding region to be expressed. Such a procedure is not applicable to a routine, v
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Faulkner James D. B.
Minton Nigel P.
Microbiological Research Authority
Robinson Douglas W.
Wai Thanda
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