Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-12-01
2003-11-25
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S326000, C530S327000, C435S069100, C930S010000
Reexamination Certificate
active
06653449
ABSTRACT:
TECHNICAL FIELD
The present invention relates to proteins having a hemolytic activity and genes encoding thereof. More specifically, the present invention relates to novel proteins having the hemolytic activity, a process for producing and the use of the same.
BACKGROUND ART
The sting injury by the jellyfish in sea bathing has occurred in various parts of the world. The sting injury by
Carybdea rastonii
or
Physalia physalis
has also occurred frequently in Japan every year in the season of sea bathing of the summertime. The degree of the symptom by sting differs by species of a jellyfish and the individual differences of patients. The first symptom is dermotoses, such as pain, flare, papule, vesicle and so on in the sting site. In a serious illness, patients may die with generating of hemorrhagic maculae and the necrosis, and also constitutional symptom, such as headache, high fever, nausea, dyspnea, and the fluctuation of a pulse.
Although such sting injury is occurring frequently, the determination and pharmacological properties of the toxic components of jellyfish have not been studied intensively.
Therefore, the development of medicines for treatment of the sting by the jellyfish is hardly performed before the present invention.
The studies on the toxic components of
Carybdea rastonii
have reported by Sato et al., and they found that there are some active substances having physiological activities, such as hemolysis, platelet agglutination, mast cell degranulation, the vessel smoothness muscle contraction, the dermal necrosis, the heart poison and the fatality in the crude extract fractions from the freeze-dried tentacle of
Carybdea rastonii
. They also examined on the platelet agglutination effect and vessel smoothness muscle contraction effect of the toxic component (Akihiko Sato, “Research on the toxic component of
Carybdea rastonii”, The Journal of the Ochanomizu Medico-dental Society
, vol. 33, No. 2, 131-151, June, 1985).
On the one hand, since the poison from the nematocyst of a jellyfish was a non-dialyzable high polymer and deactivated by treatment with acid or alkali, or by heating processing, organic solvent processing, protease processing, etc., it was thought that the main components of this poison were proteins.
Moreover, the purification of the protein toxin derived from a jellyfish has also been tried; however, the isolation and the purification of the active components maintaining the hemolytic activity were not performed since the toxin of a jellyfish itself was very easy to be deactivated. Therefore, the physical and chemical properties of the toxin from jellyfish were not known up to now.
The detailed studies on the toxic component of a jellyfish is very important for the development of drugs applying their various physiological activities, in particular, specific hemolytic activity and the platelet agglutination effect.
Therefore, the problems to be solved by the present invention is providing an approach to development of the drugs for treatment of the sting injury by the jellyfish by means of isolating the proteins or peptides having as potent hemolytic activity as possible, in the state where the physiologic activity is retained. The present invention further provides the approach to study similarities on embryology or structure, and the species specificity of the protein having hemolytic activity to evaluate the structure-activity relationship thereof.
DISCLOSURE OF THE INVENTION
The inventors extensively performed the research for isolating the proteins having the hemolytic activity from the nematocyst of
Carybdea rastonii
using the hemolytic activity as the parameter, while retaining these hemolytic activities. As the result, they found out the process for isolating and purifying the proteins retaining hemolytic activities, and found that the protein from
Carybdea rastonii
had the partial chemical structure consisting the following amino acid sequences (1)-(3), and the molecular weight of about 50,000 Da (determined by SDS gel electrophoresis).
Amino acid sequence (1):
Gly-Glu-Ile-Gln-Thr-Lys-Pro-Asp-Arg-Val-Gly-Gln-Ala-Thr (SEQ ID NO:1)
Amino acid sequence (2):
Gly-Asn-Ala-Glu-His-Val-Ala-Ser-Ala-Val-Glu-Asn-Ala-Asn-Arg-Val-Asn-Lys (SEQ ID NO:2)
Amino acid sequence (3):
Met-Ser-Asp-Gly-Phe-Tyr-Thr-Met-Glu-Asn-Ser-Asp-Arg-Arg-Lys (SEQ ID NO:3)
(wherein, an amino acid residue is written by the 3 letters notation defined by IUPAC and IUB)
Furthermore, they prepared the primers based on their partial chemical structures of the protein, and analyzed the gene sequence of about 1,000 base pair of said protein by conducting the RT-PCR on the total RNA prepared from the tentacle of
Carybdea rastonii
by using these primers. Consequently, they further determined the full primary amino acid sequence of the hemolytic active protein of
Carybdea rastonii
by means of analyzing the gene sequence at the 5′-end and 3′-end using the 5′ RACE method and 3′ RACE method.
Therefore, one embodiment of the present invention provides the specific protein having above-mentioned physiological, physical and chemical properties and represented by the amino acid (SEQ ID NO:5) or the amino acid sequence thereof partially modified by the deletion or substitution of amino acid, and/or the amino acid sequence thereof partially modified by the deletion or substitution of amino acid further one or more amino acids are added.
Another embodiment of the present invention also provides the process for preparing such proteins.
Furthermore, another embodiment provides the gene encoding such proteins, the process for preparing the specific proteins using the gene, and the drugs or the pesticides using the same.
The present invention further provides the pharmaceutical compositions or the pesticides containing the proteins using these properties, particularly, the pharmaceutical compositions having the platelet agglutination effect etc.
Moreover, since a specific antibody can also be obtained from this hemolytic active protein according to a conventional method (Cell Technology, separate volume, “Experimental protocol of antipeptide antibody”, Shujunsha Co.), the present invention also provides the pharmaceutical compositions containing said antibody.
BEST MODE FOR CARRYING OUT THE INVENTION
The isolation and purification of the proteins having the specific physiological activity provided by the present invention can specifically be performed as follows. For example, the ultrasonication of the nematocyst of
Carybdea rastonii
is carried out in phosphoric acid buffer solution, and then supernatants are collected by the centrifugal separation to obtain a crude extract. The object proteins can be separated and purified by subjecting this crude extract to ion exchange high performance liquid chromatography using TSK-GEL (Toso Co.), and the gel filtration high performance liquid chromatography with Superdex-75 (Pharmacia Co.).
The structure of the protein provided according to the present invention obtained in this way can be determined by combining the analysis procedure of the amino acid sequence by the selective degradation using the enzyme, and the analysis procedure of a gene sequence using the PCR method etc. For example, the amino acid sequence can be determined by processing the protein separated and purified as mentioned above with a lysylendopeptidase, fractionating the fragment using a high performance liquid chromatography, and analyzing it using an amino acid sequencer etc. Next, the gene sequence of the proteins can be determined by RT-PCR method etc. using the primers prepared on the basis of the amino acid sequence. Finally, the full primary amino acid sequence of the proteins can be clarified by determining the amino acid sequence on the basis of the gene sequence.
It was confirmed by such analysis that the protein provided according to the present invention has the molecular weight of about 50,000 Da (measured by SDS gel electrophoresis), and the partial amino acid sequences have the above-mentioned amino acid sequences (1) to (3).
As a r
Nagai Hiroshi
Nakajima Terumi
Crowell & Moring LLP
Low Christopher S. F.
Schnizer Holly
Suntory Limited
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