Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-16
2002-07-02
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S034000, C530S350000, C530S855000
Reexamination Certificate
active
06413729
ABSTRACT:
FIELD OF THE INVENTION
The present invention directs to peptidoglycan recognition proteins (hereinafter also briefly referred to as PGRPs), genes coding for said proteins, recombinant vectors containing said genes, transformants transformed with said recombinant vectors, a method for preparing said proteins by cultivating said transformants.
BACKGROUND OF THE INVENTION
Peptidoglycans (hereinafter also briefly referred to as PGs) are glycopeptide polymers containing N-acetylmuramic acid or N-glycolylmuramic acid and D-amino acids, and they play, as bacterial cell wall components, an important role for retention of the form of bacteria.
While endotoxins are contained only in Gram-positive bacteria, peptideglycans are contained both in Gram-negative and Gram-positive bacteria. Namely, in the Gram-negative bacteria, the peptidoglycans are contained in the cell walls to form thick layers at the outermost shells of the cell walls, and in the Gram-positive bacteria, they are contained in the cell walls to form thin layers inside outer membranes of the cell walls. This means that almost all procaryotes contain peptidoglycans in their cell walls, only exception being archaebacteria (such as methane bacteria and high acidophil thermophiles) which contain no endotoxins nor peptidoglycan. On the sharp contrary, eucaryotes such as mammals contain no peptidoglycans in their cell walls.
Thus peptidoglycans can be said as an useful indicator for existence of bacteria in various kinds of objects.
Accordingly, a trace amount of bacteria contained in various kinds of objects can be detected by subjecting the objects to detection and measurement of peptidoglycans. The detection and measurement of peptidoglycans are therefore expected to be applied to safety tests of drugs, microbial tests of water and food, and diagnoses of infectious diseases.
The chemical structures of peptidoglycans are classified to into several kinds although they vary with bacteria species. For example, peptide sub-units each consisting of 3 or 4 amino acids are attached through the carboxyl groups of muramic acid molecules to a saccharide chain having the repeating structure of N-acetylglucosamine and N-acetylmuramic acid which are linked by a &bgr;-1,4 bond to each other, and the peptide sub-units are crosslinked directly or through other peptides, thereby forming a network structure in a bag form as a whole.
The peptidoglycans have various biological activities. Examples thereof in vitro include various functions to immune response cells such as macrophages, B lymphocytes and T lymphocytes, destruction of blood platelets, growth enhancement of fibroblasts, enhancement of bone resorption and activation of complements. Examples thereof in vivo include enhancement or inhibition of humoral immune responses, enhansment of cellular immunity, stimulation of cell endothelial systems, transient leukopenia and subsequent hypercytosis, enhancing the functions of interferon inducing factors, potentiation of natural resistance, induction of experimental autoimmune diseases, pyrogenic functions, enhancing sensitivity to the toxicity of endotoxins, enhancement or inhibition of sleep, formation of epithelioid granulomas, functions of inducing hemorrhagic necrotics at sites treated with tubercle bacillus, and acute or chronic toxicity. Many of these activities are in common with the functions of endotoxins, and weaker in intensity than the endotoxins. However, detection and measurement of peptidoglycans contained, for example, in drugs or food are considered to become increasingly important from now on, because of such activities of the peptidoglycans.
As a method for detecting and measuring peptidoglycans, a method developed by the present inventors in which a silkworm hemolymph-derived reagent is used is reported in Japanese Examined Patent Publication No. 7-114707. According to this method, it is possible to measure the total PG content in a sample. However, it is impossible, for example, to test the PG distribution in a solid such as a tissue section, or to conduct a specific stain of bacteria in a tissue section.
Accordingly, the development of a method for specifically detecting and measuring PGs which can be used for such purposes has been desired.
Such a method for specifically detecting and measuring PGs has been considered to be accomplished by using substances specifically binding to the PGs. However, no substance having preferable properties is available at low cost in large amounts, so that no practical method has been developed yet.
Namely, as the substances specifically binding to the PGs, for example, lysozyme and the peptidoglycan recognition protein discovered by the present inventors [
J. Bio. Chem
. 271 (23), 13854-13860 (1992)] are known. However, lysozyme is an enzyme which decomposes the PGs by binding to them, so that it is unsuitable for use for such purposes. On the other hand, the peptidoglycan recognition protein is only obtained by purification from hemolymph of insects such as silkworms, in which it is contained in very small amounts. In respect to cost, therefore, it is hard to say that a reagent using this peptidoglycan recognition protein is practical.
Accordingly, the development of a method for obtaining the peptidoglycan recognition proteins at low cost in large amounts has been desired.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for obtaining peptidoglycan recognition proteins in large amounts at high purity.
As a result of intensive investigation for attaining the above-mentioned object, the present inventors have succeeded in elucidating the primary structure of PGRPs to clone cDNA coding for PGRPs, determining amino acid sequences of the PGRPs based on a nucleotide sequence of the cDNA, and producing the PGRPs by recombinant technology using the DNA, thereby attaining the above-mentioned object.
That is, the present invention provides:
(1) A recombinant protein comprising the following amino
acid sequence (SEQ ID NO: 1):
Asp Cys Asp Val Val Ser Lys Lys Gln Trp Asp Gly Leu Ile Pro Val
His Val Ser Tyr Leu Ala Arg Pro Val Ser Leu Val Ile Val Gln His
Thr Val Thr Pro Phe Cys Arg Thr Asp Ala Gly Cys Glu Glu Leu Val
Arg Asn Ile Gln Thr Asn His Met Glu Ala Leu Gln Tyr Trp Asp Ile
Gly Pro Ser Phe Leu Val Gly Gly Asn Gly Lys Val Tyr Glu Gly Ser
Gly Trp Leu His Val Gly Ala His Thr Tyr Gly Tyr Asn Ser Arg Ser
Ile Gly Val Ala Phe Ile Gly Asn Phe Asn Thr Asp Glu Pro Ser Gly
Ala Met Leu Glu Ala Leu Arg Ser Leu Leu Arg Cys Gly Val Glu Arg
Gly His Leu Ala Gly Asp Tyr Arg Val Val Ala His Arg Gln Leu Ile
Ala Ser Glu Ser Pro Gly Arg Lys Leu Tyr Asn Gln Ile Arg Arg Trp
Pro Glu Trp Leu Glu Asn Val Asp Ser IIe Lys Asn Ala;
(2) the recombinant protein according to (1), wherein the
recombinant protein has peptidoglycan recognition activity and
has the following amino acid sequence (SEQ ID NO:1) or one
obtained by deletion, substitution or addition of one or more
amino acids in the following amino acid sequence (SEQ ID NO:1):
Asp Cys Asp Val Val Ser Lys Lys Gln Trp Asp Gly Leu Ile Pro Val
His Val Ser Tyr Leu Ala Arg Pro Val Ser Leu Val Ile Val Gln His
Thr Val Thr Pro Phe Cys Arg Thr Asp Ala Gly Cys Glu Glu Leu Val
Arg Asn Ile Gln Thr Asn His Met Glu Ala Leu Gln Tyr Trp Asp Ile
Gly Pro Ser Phe Leu Val Gly Gly Asn Gly Lys Val Tyr Glu Gly Ser
Gly Trp Leu His Val Gly Ala His Thr Tyr Gly Tyr Asn Ser Arg Ser
Ile Gly Val Ala Phe Ile Gly Asn Phe Asn Thr Asp Glu Pro Ser Gly
Ala Met Leu Glu Ala Leu Arg Ser Leu Leu Arg Cys Gly Val Glu Arg
Gly His Leu Ala Gly Asp Tyr Arg Val Val Ala His Arg Gln Leu Ile
Ala Ser Glu Ser Pro Gly Arg Lys Leu Tyr Asn Gln Ile Arg Arg Trp
Pro Glu Trp Leu Glu Asn Val Asp Ser Ile Lys Asn Ala;
(3) a gene coding for a protein comprising the following amino
acid sequence (SEQ ID NO:1):
Asp Cys Asp Val Val Ser Lys Lys Gln Trp Asp Gly Leu Ile Pro Val
His Val Ser Tyr Leu Ala Arg Pro Val Ser Leu Val Ile Val Gln His
Thr Val Thr Pro Phe Cys Arg Thr Asp Ala Gly Cys Glu Glu Leu Val
Arg Asn Ile Gln Thr Asn His
Ashida Masaaki
Ochiai Masanori
Tsuchiya Masakazu
Colin David G.
Dike Bronstein Roberts & Cushman IP Group
Lowan Cara Z.
Romeo David S.
Wako Pure Chemical Industries, Inc.
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