Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Cyclic peptides
Patent
1989-04-19
1991-11-26
Lee, Lester L.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Cyclic peptides
A61K 3702, C07K 708
Patent
active
050683144
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a novel polypeptide and a method for preparing the same, more particularly to a novel polypeptide exhibiting strong affinity for lipopolysaccharide (endotoxin) and a method for preparing the same.
BACKGROUND ART
Endotoxin is also called an intracellular toxin, which term refers comprehensively to toxic substances existing in the cells of Gram-negative bacteria. The components of endotoxin are lipopolysaccharides (hereinafter called "LPS").
In the prior art, the pyrolysis method, the ultrafiltration method, and the affinity chromatographic method with polymixin B are known methods for removing endotoxin.
However, the pyrolysis method is a method which thermally decomposes LPS through a dry heat treatment at 250.degree. C. or higher to remove LPS from glass vessels, etc. by decomposition. This pyrolysis method cannot be utilized for separating LPS from a substance which is unstable to heat. The ultrafiltration method is effective for separation of LPS from low molecular weight substances, but it is not applicable in principle for separation of endotoxin from high molecular weight substances. The affinity chromatographic method with polymixin B may be expected to be practically applied from the point of utilizing the affinity possessed by polymixin B for LPS, but use is limited because of the toxicity of polymixin B and thus, this method has not been presently practically applied.
Thus, there has not been found yet a practically effective method as the method for separating effectively and stably LPS from among high molecular weight physiologically active substances.
Accordingly, the present inventors have intensively studied in order to find novel substances exhibiting affinity for LPS. In the present invention, this substance is called as LPS-binding polypeptide (sometimes abbreviated to LBP). Consequently, they successfully extracted and isolated a novel polypeptide from horseshoe crab hemocyte, synthesized this polypeptide by the synthetic method such as solid phase peptide synthesis, and further found that said polypeptide exhibits strong affinity for LPS and has biological activities such as antibacterial activity and blastgenesis inhibition action, etc., thus accomplishing the present invention.
DISCLOSURE OF THE INVENTION
The present invention concerns a polypeptide represented by the formula: ##STR2## wherein Lys represents lysine, Trp tryptophan, Cys cystine, Phe phenylalanine, Arg arginine, Val valine, Tyr tyrosine, Gly glycine, Ile isoleucine and X a hydroxyl group or an amino group, its analogue and a method for preparing said polypeptide.
The compound of the present invention is a polypeptide comprising 17 amino acids, in which the carboxyl group of arginine which is the C-end amino acid is amidated under the extracted or isolated condition. Even when this polypeptide is converted to an acid by hydrolysis, the affinity for LPS remains high.
The polypeptide of the present invention can be extracted and isolated from horseshoe crab hemocyte of Tachypleus tridentatus, Tachypleus gigas or Limulus Tachypleus gigas as described below.
More specifically, the residue after hypotonic extraction of hemocyte of Tachypleus tridentatus is extracted under acidic condition, for example, in diluted mineral acid such as hydrochloric acid, nitric acid, sulfuric acid, etc; or in organic acid, for example, low aliphatic acid such as acetic acid, etc. The extract obtained is subjected to purification means such as gel filtration, chromatography, etc., whereby the polypeptide can be isolated.
The polypeptide of the present invention can be produced by the synthetic methods such as the peptide synthesis method, e.g. solid phase peptide synthesis method, liquid phase peptide synthesis method, etc.
More specifically, for example, in the solid phase synthetic method after the carboxyl group of N-protected arginine is bonded to an insoluble resin having amino groups, sometimes through a spacer having both carboxyl group and a functional group capable of bonding to a carboxy
REFERENCES:
patent: 4663435 (1987-05-01), Brady
Tokunaga, et al., "Tachyplesin a Class of Antimicrobial Peptide from the Hemocytes of the Horseshoe Crab (Tachypleus tridentatus), J. Biol. Chem., v"ol. 263, No. 32, pp. 16709-16713, p. 1988.
Morita, et al., "Isolation and Biological Activities of Limulus Anticoagulant which Intera ts with Lipopolysaccharide", J. Biol. chem. 97, 1611-1620, 1985.
Shieh, et al., "Synthesis and properties of tachyplesin I, a lipolysaccharide-binding peptide, from Tachypleus tridentatus" Feb, 252, pp. 121-124.
Aketagawa et al., "Primary Structure of Limulus Anticoagulant Anti-lipopolysaccharide Factor", J. Biol. Chem. vol. 261, pp. 7357-7365, 1986.
Iwanaga Sadaaki
Miyazaki Kyosuke
Nakamura Takanori
Ohno Motonori
Bierman Jordan B.
Lee Lester L.
Marshall S. G.
Seikagaku Kogyo Co. Ltd.
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