T7 promoter-based expression system

Details T7 promoter-based expression system T7 promoter-based expression system

2000-04-03

2003-03-25

Yucel, Remy (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...

C435S320100, C435S252300, C435S471000, C435S325000, C435S455000, C536S023100, C536S024100

Reexamination Certificate

active

06537779

ABSTRACT:

The invention relates to expression systems for the recombinant synthesis of polypeptides, in particular to T7 promoter-driven protein expression systems. The invention also relates to expression vectors for use in such systems.
A large number of mammalian, yeast and bacterial host expression systems are known (Methods in Enzymology (1990), 185, Editor: D. V. Goeddel). Of particular interest are those which use T7 RNA polymerase. The ability of T7 RNA polymerase and equivalent RNA polymerases from T7-like phages to transcribe selectively any DNA that is linked to an appropriate promoter can serve as the basis for a very specific and efficient production of desired RNAs both in vitro and inside a cell.
U.S. Pat. No. 4952496 (Studier) discloses a process whereby T7 RNA polymerase can be expressed and used to direct the production of specific proteins, all within a host
E. coli
cell. Specific proteins of interest include antigens for vaccines, hormones, enzymes, or other proteins of medical or commercial value. Potentially, the selectivity and efficiency of the phage RNA polymerase could make such production very efficient. Furthermore, the unique properties of these phage RNA polymerases may make it possible for them to direct efficient expression of genes that are expressed only inefficiently or not at all by other RNA polymerases. These phage polymerases have the further advantage that it is possible to selectively inhibit the host cell RNA polymerase so that all transcription in the cell will be due to the phage RNA polymerase.
An expression system based on the above is now commercially available. This is the pET system obtainable from Novagen Inc. 597 Science Drive, Madison, WI 53711. This system is suitable for the cloning and expression of recombinant proteins in
E. coli
. See also Moffat et al, J. Mol. Biol., 1986, 189, 113-130; Rosenberg et al, Gene, 56, 125-135; and Studier et al, Meth. Enzymol. 1990, 185, 60-89.
However, despite the provision of the pET system, there remains the need for further, improved T7 promoter-driven expression systems.
We have now devised such a system which provides improved control of expression and improved levels of protein expression, when compared to available 17-based expression systems. We provide a T7 promoter-driven expression system wherein basal expression in the absence of inducer is reduced to a level which permits the cloning and expression of toxic gene products not possible with currently available T7 based expression systems whilst not influencing induced productivity. Moreover, our present invention also allows control of production of heterologous proteins in an inducer concentration-dependent manner over a wide range of expression levels so that an optimum level of expression can be identified. This level of control over expression and production of heterologous protein is not possible with currently available T7 based expression systems.
Therefore in a first aspect of the invention we provide a T7 promoter-driven protein expression system comprising an operator sequence downstream of the T7 promoter sequence, and having a further operator sequence upstream of the T7 promoter sequence.
We have found that the further operator is preferably a native lac operator (lacO) sequence. Or a perfect palindrome operator (ppop) sequence. More preferably the native (lac) operator sequence downstream of the T7 promoter sequence is replaced by a ppop sequence, so as to provide a tandem ppop operator.
The T7 promoter driven expression system is conveniently constructed as follows- The target gene of interest is cloned in a plasmid under control of bacteriophage transcription and translation signals. The target gene is initially cloned using a host such as
E.coli
DH5&agr;, HB101 that does not contain the T7 RNA polymerase gene. Once established, plasmids are transferred into expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under for example lac UV5 control. Other convenient promoters include lac, trp, tac, trc, and bacteriophage &lgr; promoters such as pL and pR. Expression is then induced by the addition of an inducer such as IPTG (isopropyl-&bgr;-D-1-thiogalactopyranoside), lactose or melibiose. Other inducers may be used and are described more fully elsewhere. See The Operon, eds Miller and Reznikoff (1978). Inducers may be used individually or in combination.
The plasmid preferably includes one or more of the following: a selectable antibiotic resistance sequence, a cer stability element, and a multiple cloning site. The construction of appropriate plasmids will be apparent to the scientist of ordinary skill. Examples of preferred plasmids comprising one or more of the above features are illustrated by the pZT7#3-series of plasmids in the accompanying Figures. These were constructed starting from a vector pZEN0042 disclosed (as pICI0042) in our European Patent Application No. 0 502 637 (ICI). The 3-series plasmids of this invention include pZT7#3.0, pZT7#3.1, pZT7#3.2 and pZT7#3.3. A particularly preferred plasmid of this invention is the pZT7#3.3 plasmid.
The chromosomal copy of the T7 RNA polymerase gene, for example under lac UV5 control, is preferably introduced into the host cells via the &lgr; bacteriophage construct, &lgr;DE3, obtainable from Novagen. The T7 RNA polymerase expression cassette may also be delivered to the cell by infection with a specialised bacteriophage &lgr; transducing phage that carries the gene (CE6, U.S. Pat. No. 4,952, 496.
Compatible plasmids such as pLysS and pLysE (also available from Novagen) may also be introduced into the expression host. These plasmids encode T7 lysozyme, which is a natural and selective inhibitor of T7 RNA polymerase, and thus reduces its ability to transcribe target genes in uninduced cells. pLysS hosts produce low amounts of T7 lysozyme, while pLysE hosts produce much more enzyme and therefore provide more stringent control.
Any convenient compatible prokaryotic or eukaryotic host cell may be used. The most commonly used prokaryotic hosts are strains of
E.coli
, although other prokaryotic hosts such as
Salmonella typhimurium, Serratia marsescens, Bacillus subtilis
or
Pseudomonas aeruginosa
may also be used. Mammalian (e.g. Chinese hamster ovary cells) or other eukaryotic host cells such as those of yeast (e.g.
Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces poinbe
or
Kluyveroromyces lactis
), filamentous fungi, plant, insect, amphibian or ovarian species may also be useful. A particular host organism is a bacterium, preferably
E. coli
(e.g. K12 or B strains).
Any convenient growth medium may be used depending on the host organism used. For
E.coli
, practice of this invention includes, but is not limited to complex growth media such as L-broth or minimal growth media such as M9 (described hereinafter).
The invention will now be illustrated but not limited by reference to the following detailed description Examples, Tables and Figures wherein:
Table 1 gives details of plasmids expressing h-TNF&agr; used in the Examples. Tables 2-4 gives details of vectors used in the Examples and their relative performance.
Table 5 gives details of the composition of M9 minimal growth medium.
Table 6 gives details of h-TNF&agr; expression in various growth media.
Table 7 gives details of host/transformation efficiencies for vectors used in the Examples.
Table 8 gives details of DNase 1 productivity in conjunction with the pZT7#3.3:DNase 1 vector.
Tables 9-11 give details of accumulation levels for LAR d1 (aa1275-1623), ZAP70 (4-260) 6HIS and MCP-1 {9-76} used in the Examples.
Table 12 shows the sequences of oligonucleotides used in the construction of pZT7#3.3 and intermediate vectors.
Table 13 shows the nucleic acid sequence of hTNF&agr;.
Table 14 shows the ZAP70 (4-260) 6HIS nucleic acid sequence.
Table 15 shows the LAR d1 (aa1275-1623) nucleic acid sequence.
Table 16 shows the bovine pancreatic DNase 1 nucleic acid sequence.
Table 17 shows the human carboxypeptidase B (muta

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