Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-10-21
2001-07-03
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S199000, C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06255081
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a thermostable Flap endonuclease effective for genetic recombination and genetic shuffling based on low homology, as well as to a gene thereof.
2. Description of the Prior Art
There are a method for random mutation of a broad region in a gene and a method for random mutation of a local region in a gene. In the former method, polymerase chain reaction (PCR) is applied such that a specific nucleotide is deleted to induce mutation at the time of replication of the target gene, and in the latter method, PCR using mixed primers is applied to mutate the target site. However, there is still no method for highly efficient induction of genetic recombination or genetic shuffling based on low homology in vitro. A speculative mechanism of genetic recombination and genetic shuffling in vivo is shown in FIG.
1
. Step 1 of
FIG. 1
shows formation of a single-stranded overhang by a 3′-5′ exonuclease; step 2 shows formation of temporary nucleotide base pairs based on low homology; step 3 shows repair of the gap by DNA polymerase and formation of a Flap structure; step 4 shows removal of the Flap single strand by a Flap endonuclease; and step 5 shows ligation of nicks by a DNA ligase. However, the properties of the enzymes catalyzing the respective steps are not fully elucidated. The Flap endonuclease is an enzyme catalyzing step 4. As shown in
FIG. 2
, the Flap endonuclease specifically recognizes the Flap structure in DNA and cleaves the single strand (called Flap), and this enzyme is found in mammalian cells and yeast cells. Since its origin is organisms living at normal temperatures, this enzyme has poor thermostability and thus is not suitable for artificial genetic shuffling reaction, including PCR.
Because the conventional Flap endonuclease is unstable at high temperatures, it cannot be used to develop methods where genetic recombination or genetic shuffling based on low homology in vitro is induced at high temperatures. However, if a thermostable Flap endonuclease functioning stably at high temperatures can be obtained, it becomes possible to develop new techniques of conducting artificial homologous recombination or genetic shuffling highly efficiently by coupling the enzyme reaction with PCR. Accordingly, development of the thermostable Flap endonuclease stable at high temperature has long been desired.
SUMMARY OF THE INVENTION
Under these circumstances, the object of the present invention is to provide a novel thermostable Flap endonuclease and a gene thereof.
To achieve this object, the present inventors directed their attention to a hyperthermophile bacterium growing at 90 to 100° C., and as a result, a gene presumed to bring about the activity of the present enzyme was found in a gene sequence from the bacterium. Further, the enzyme was produced in
E. coli
by use of said gene, and it was confirmed that this enzyme is stable at high temperatures (75° C. or more) and exhibits structurally specific endonuclease activity. The present invention was thereby completed.
That is, the present invention relates to a thermostable Flap endonuclease whose optimum temperature is 75° C. or more.
Further, the present invention relates to a DNA coding for the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; and (b) a protein with Flap endonuclease activity, consisting of an amino acid sequence where in the amino acid sequence (a), one or more amino acids are deleted, substituted or added.
The DNA is a DNA specifically shown in SEQ ID NO:1.
The addition, deletion or substitution of amino acids can be effected using site-directed mutagenesis known in the art (see e.g. Nucleic Acid Research, Vol. 10, No. 20, pp. 6487-6500 (1982)). The number of one or more amino acids added, deleted or substituted is the number of amino acids which can be added, deleted or substituted by site-directed mutagenesis.
Further, the present invention relates to a recombinant vector comprising said DNA.
Further, the present invention relates to a transformant transformed with the recombinant vector comprising said DNA.
Further, the present invention relates to a process for producing a thermostable Flap endonuclease, which comprises culturing said transformant in a medium to produce the thermostable Flap endonuclease.
REFERENCES:
patent: 5843669 (1998-12-01), Kaiser et al.
patent: 5846717 (1998-12-01), Brow et al.
patent: 5874283 (1999-02-01), Harrington et al.
patent: 9-239440 (1997-04-01), None
Ishikawa Kazuhiko
Kawasaki Satoko
Kosugi Yoshitsugu
Matsui Eriko
Matsui Ikuo
Director - General of Agency of Industrial Science and Technolog
Foley & Lardner
Patterson Jr. Charles L.
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