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
1999-10-20
2001-07-10
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S252300, C435S320100, C530S350000
Reexamination Certificate
active
06258561
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of producing a 19P2 ligand (19P2L) or an amide thereof or a salt thereof which comprises preparing a fusion protein or peptide and subjecting said fusion protein or peptide to a peptide bond cleavage reaction.
BACKGROUND OF THE INVENTION
In the production of a protein or peptide by recombinant DNA technology, it is more or less common practice to have the protein or peptide expressed in the form of a fusion protein in view of the liability of the protein or peptide to be decomposed within living cells. For excision of the objective protein or peptide from the fusion protein, a chemical method for cleavage using cyanogen bromide (Itakura et al., Science, 198, 1056, 1977) and an enzymatic method using factor Xa (Nagai et al., Methods in Enzymology, 153, 46, 1987) are known.
Furthermore, as a method for cleavage of a peptide bond in a protein, cleavage of the acylcysteine bond with 2-nitro-5-thiocyanobenzoic acid is known [Seikagaku Jikken Koza 1, Tanpakushitsu-no-Kagaku II (Biochemical Experiment Series 1, Protein Chemistry II), Japanese Society of Biochemistry (ed.), Tokyo Kagaku Dojin, 247-250, 1976]. However, there is no disclosure on the excision of an objective protein or peptide from a protein.
The prior art method which involves use of cyanogen bromide cannot be applied to the production of methionine-containing peptides, while the method involving use of factor Xa has drawbacks, for example in terms of excision yield.
Therefore, a demand exists for a technology by which an objective protein or peptide may be efficiently excised from a fusion protein or peptide.
The inventors of the present invention explored in earnest for a technology by which the novel bioactive peptide 19P2 ligand (19P2L, which is named as a “prolactin-releasing peptide (PrRP)” in Hinuma et al., Nature 393, 272-276, (1998)) may be produced with high efficiency and found that 19P2L can be efficiently produced by preparing a fusion protein or peptide comprising 19P2L fused to a protein or peptide having cysteine at its N-terminus and subjecting the fusion protein or peptide to a peptide bond cleavage reaction. The inventors did further research on the basis of the above finding and have accomplished the present invention.
SUMMARY OF THE INVENTION
The present invention, therefore, is directed to:
(1) A method of producing a 19P2L or an amide thereof or a salt thereof which comprises subjecting a fusion protein or peptide comprising the 19P2L fused to a protein or a peptide having a cysteine residue at the N-terminus to a reaction for cleavage of the peptide bond on the amino terminal side of the of the cysteine residue,
(2) A method of producing a 19P2L or an amide thereof or a salt thereof which comprises
{circle around (1)} culturing a transformant harboring a vector containing a gene coding for a fusion protein or peptide comprising a 19P2L fused to a protein or a peptide having a cysteine residue at the N-terminus to express the fusion protein or peptide and
{circle around (2)} subjecting the fusion protein or peptide expressed to a reaction for cleavage of the peptide bond on the amino-terminal side of the cysteine residue,
(3) The method of the above item (1) or (2) wherein the reaction for cleavage of the peptide bond on the amino-terminal side of the cysteine residue comprises {circle around (1)} cyanylation followed by {circle around (2)} ammonolysis or hydrolysis,
(4) The method of the above item (1) or (2) wherein the reaction for cleavage of the peptide bond on the amino-terminal side of the cysteine residue comprises {circle around (1)} cyanylation followed by {circle around (2)} ammonolysis to produce the amide of 19P2L,
(5) The method of the above item (1) or (2) wherein the 19P2L is bovine 19P2L (SEQ ID NO:7), rat 19P2L (SEQ ID NO:8), or human 19P2L (SEQ ID NO:9),
(6) A fusion protein or peptide comprising a 19P2L fused to a protein or a peptide having a cysteine residue at its N-terminus,
(7) A vector containing a gene coding for the fusion protein or peptide according to the above item (6), and
(8) A transformant harboring the vector according to the above item (7).
REFERENCES:
patent: 0 499 990 A2 (1992-08-01), None
patent: 97/08317 (1997-03-01), None
patent: 97/24436 (1997-07-01), None
Nobuyuki Koyama, et al. “A Novel Procedure For The Preparation Of Biologically Active Recombinant Peptides Using a Cyanylation Reaction”, Journal of Biotechnology, vol. 32, No. 2, 1994, pp. 273-281.
Shuji Hinuma, et al. “A Prolactin-Releasing Peptide In The Brain”, Nature, vol. 393, May 21, 1998, pp. 272-275.
Copy of Communication dated Nov. 27, 1998 with European Search Report re European Pat. Appln. No. 98111725.2-2106.
Masato Suenaga
Osamu Nishimura
Takeo Moriya
Yoko Tanaka
Buchanan Robert L.
Conlin David G.
Edwards & Angell LLP
Low Christopher S. F.
Srivastava Devesh
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