Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-11-10
2000-12-26
Yucel, Remy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435471, 435476, 435478, 4353201, C12P 2106
Patent
active
061657492
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a plasmid vector usable in genetic recombinant technology and to a method for expressing a gene by using the plasmid vector. The present invention also relates to a method for isolating a desired gene by using such a plasmid vector. In addition, the present invention relates to a restriction enzyme and a gene thereof available as a genetic engineering reagent, in more detail to the AccIII restriction endonuclease and a DNA coding therefor.
BACKGROUND ART
In constructing an expression system for a desired gene by genetic recombinant technology, expression of the gene is controlled by bringing it under control of a promoter recognized by the RNA polymerase of the host used. In the case of a gene encoding a protein harmful to the host, however, plasmid construction itself is sometimes hampered by expression of the product of the gene due to the inability to stringently control the expression of the promoter used.
As an expression system resolving that problem, the pET system (produced by Novagen) has been developed, which uses the RNA polymerase of the bacteriophage T7, which infects Escherichia coli, with Escherichia coli as a host [Journal of Molecular Biology, Vol. 189, pp. 113-130 (1986); Gene, Vol. 56, pp. 125-135 (1987)]. The pET system is a system that allows T7 RNA polymerase, which has high promoter recognition specificity and high transcription activity, to be expressed in Escherichia coli, which T7 RNA polymerase transcribes a desired gene placed downstream of the T7 promoter on an expression vector and causes high expression of the gene. Because transcription of the desired gene occurs in the presence of T7 RNA polymerase, plasmid construction in the host is possible without expressing the desired gene, provided that the host does not produce the polymerase; plasmid construction itself is never hampered, as in cases where the expression system is constructed, while the desired gene is kept under control of a promoter recognized by the RNA polymerase of the host.
However, because the T7 RNA polymerase gene has been cloned onto the .lambda.-phage vector and lysogenized into the expression host, there is no freedom of host choice; painstaking procedures are needed if the host is changed. In addition, because the expression of T7 RNA polymerase in the host is not stringently controlled, T7 RNA polymerase is expressed even when the host is in a non-inductive condition, resulting in expression of the desired gene placed downstream of the T7 promoter on the expression vector even in a non-inductive condition. To suppress such expression of the desired gene in a non-inductive condition, T7 RNA polymerase activity is inhibited using T7 lysozyme, a T7 RNA polymerase inhibitor [Journal of Molecular Biology, Vol. 219, pp. 37-44 (1991)], or T7 RNA polymerase is prevented from getting access to the T7 promoter by placing a lactose operator downstream of the T7 promoter [Journal of Molecular Biology, Vol. 219, pp. 45-59 (1991)].
However, even these countermeasures are unsatisfactory in terms of effect against T7 RNA polymerase of high transcription activity so that the activity of T7 RNA polymerase in a non-inductive condition cannot be completely inhibited. For this reason, if the desired gene product is lethal to the host, it is impossible in some cases to prepare a transformant for expression of the gene, even when plasmid construction is possible. In other words, the pET system involves two problems to be resolved: one of the inability to freely change the host, and the other of inaccurate control of T7 RNA polymerase expression.
On the other hand, there is a bacteriophage having characteristics similar to those of the bacteriophage T7, known as the bacteriophage SP6 [Science, Vol. 133, pp. 2069-2070 (1961)], which infects Salmonella typhimurium. The RNA polymerase produced by the bacteriophage SP6, a single peptide having a molecular weight of about 100,000, is commonly used for in vitro RNA synthesis since it possesses high promoter recognition specifici
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Kato Ikunoshin
Oshima Atsushi
Sagawa Hiroaki
Ueno Harumi
Takara Shuzo Co. Ltd.
Yucel Remy
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