Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-12-27
2003-08-05
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023400
Reexamination Certificate
active
06602689
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to expression of a fused protein, more specifically to a fused DNA sequence including a DNA sequence coding a heat-resistant protein, a fused protein expressed by said fused DNA sequence, and a method for expressing said fused protein.
Progress in genetic engineering has enabled analysis of a protein which has been purified from a natural substance, at a genetic level and artificial amplification of a desired protein (Itakura et al., Science, vol. 198, p. 1056 (1977)). By application of a DNA sequence to which thio-redoxin (hereinafter referred to as “TRX” in the specification) (International Provisional Patent Publication No. 507209/1993) or glutathione-S-transferase (hereinafter referred to as “GST” in the specification) (International Provisional Patent Publication No. 503441/1989) which has been invented thereafter is fused, even a protein which is inherently expressed with difficulty can be expressed, and a technique of expressing a fused protein has been used widely.
TRX and GST can be applied to fusion and expression of various proteins which are expressed with difficulty, but even in GST which has been essentially used for the purpose of expressing a soluble fused protein, a fused protein becomes insoluble depending on a protein to be fused so that productivity is lowered, or a fused protein to which TRX is fused may have a problem that a nonspecific reaction is liable to occur. Therefore, it has been desired to provide a fused protein having further excellent operatability and productivity.
SUMMARY OF THE INVENTION
Thus, an object of the present invention is to provide a novel fused DNA sequence having excellent operatability and productivity for expressing a desired protein or peptide, a fused protein expressed from said fused DNA sequence, and a method for expressing the fused protein using said fused DNA sequence.
The present inventors have studied intensively in order to solve the problems in the art and consequently found that when a DNA sequence coding a selected protein or peptide and a DNA sequence coding a heat-resistant protein are fused directly or indirectly and a fused protein is expressed from the resulting fused DNA sequence, the productivity of the desired protein or peptide is raised, and said fused protein has heat resistance to make a purification step simple and easy, to accomplish the present invention.
That is, the present invention relates to a fused DNA sequence comprising a DNA sequence coding a heat-resistant protein or peptide, fused directly or indirectly to a DNA sequence coding a selected protein or peptide, a fused protein expressed by said fused DNA sequence, and a method for expressing the fused protein using said fuse-DNA sequence.
The fused protein of the present invention has high solubility and can maintain even heat resistance derived from heat-resistant protein genes. Because of such a characteristic of the fused protein, when the fused protein is purified, unnecessary substances can be removed simply and easily by heat treatment so that the fused protein can be obtained with good yield.
In the case of TRX derived from
Escherichia coli
and GST derived from
Schistosoma japonicum
, which have been widely used as a fused protein,
Escherichia coli
and
Schistosoma japonicum
can live in bodies of mammals and other creatures so that when a fused protein using TRX or GST is used as an antigen of an immunoreaction, a nonspecific reaction due to
Escherichia coli
or
Schistosoma japonicum
might be caused. To the contrary, the great characteristic of the fused protein of the present invention resides in that a heat-resistant protein derived from a thermophilic bacterium which cannot live in living bodies of mammals and other creatures is used so that even when the fused protein of the present invention is used as an antigen of an immunoreaction, a nonspecific reaction derived from the fused protein is caused with difficulty.
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patent: 4797359 (1989-01-01), Finkelstein
patent: 5723588 (1998-03-01), Donofrio et al.
patent: 6280998 (2001-08-01), Mathur et al.
patent: WO 87/05935 (1987-10-01), None
Kath et al., “Identification, Cloning, and Expression of the Gene for Adenylate Kinase from the Thermoacidophilic Archaebacterium Sulfolobus acidocaldarius”, Archives of Biochemistry and Biophysics vol. 307 No. 2 (Dec. 1993) pp. 405-410.*
Darimont et al., “Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima”, The EMBO Journal vol. 13 No. 8 (1994) pp. 1772-1781.*
Parsons, J.A. et al. “Peptide Hormones”, published by University Park Press (Baltimore), see Rudinger et al. Chapter 1, pp. 1-6, Jun. 1976.*
Lazar et al. Mol. Cell Biol. 8(3):1247, Mar. 1988.*
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Smith et al. Gene 67:31, Jul. 1988.*
Andreas Heltzel, et al., Journal of Bacteriology, vol. 176, No. 15, pp. 4790-4793, Aug. 1994, “Cloning, Expression, and Molecular Characterization of the Gene Encoding an Extremely Thermostable [4Fe-4s] Ferredoxin from the Hyperthermophilic Archaeon Pyrococcus Furiosus”.
Thomas Kath, et al., Archives of Biochemistry and Biophysics, vol. 307, No. 02, pp. 405-410, Dec. 1993, “Identifiication, Cloning, and Expression of the Gene for Adenylate Kinase from the Thermoacidophilic Archaebacterium Sulfolobus Acidocaldarius”.
M. Moracci, et al., Carbohydrate Bioengineering, pp. 77-84, 1995, “Properties and Production of the &bgr;-Glycosidase from the Thermophilic Archaeon Sulfolobus Solfataricus Expressed in Mesophilic Hosts”.
Fujii Nobuyuki
Okada Masahisa
Ueno Eiichi
Allen Marianne P.
Fujirebio Inc.
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