Endotoxin binding and neutralizing protein and uses thereof
OwnerNorman R. Wainwright, Thomas J. Novitskyinventors
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Abstract
Endotoxin binding/neutralizing proteins capable of binding endotoxin in vivo, thereby neutralizing the toxic effect or bioactivity of endotoxin which are isolated from a horseshoe crab such as Limulus polyphemus, pharmaceutical compositions and pharmaceutical uses of the proteins, a method of purifying the proteins and an assay for endotoxin based on the proteins, are disclosed.
Claims81
A method for isolating an endotoxin binding protein from a horseshoe crab, which comprises:
- subjecting amebocytes obtained from a horseshoe crab to hypotonic shock to lyse said amebocytes, and obtain cell debris from said lysed amebocytes,
- extracting said cell debris with a solution containing a denaturant selected from the group consisting of urea and guanidine hydrochloride, to produce an extract,
- passing said extract through a first ultrafiltration membrane having a molecular cutoff of from 20,000 to 50,000 daltons, to obtain a filtrate,
- concentrating said filtrate by passing it through a second ultrafiltration membrane having a molecular cutoff of from 5,000 to 10,000, to produce a retentate,
- subjecting said-retentate to cation exchange chromatography at a pH of from about 5 to 6, using an elution buffer which comprises urea, and eluting a solution containing a peak of endotoxin binding activity,
- applying said solution containing said peak of endotoxin binding activity to a reverse phase column, and adding a buffer to said reverse phase column, to obtain a solution containing purified endotoxin binding protein.
A method for ameliorating the biological effects of endotoxin in vivo, which comprises administering to a mammal in need of such treatment an effective amount of an endotoxin-binding protein of the horseshoe crab.
A method for ameliorating the biological effects of endotoxin in vivo, which comprises administering to a mammal in need of such treatment an effective amount of an endotoxin-binding protein preparation obtained from the horseshoe crab according to the following process:
- (a) subjecting amebocytes obtained from a horse-shoe crab to hypotonic shock to lyse said amebocytes, and obtain cell debris from said lysed amebocytes;
- (b) extracting said cell debris with a solution containing a denaturant selected from the group consisting of urea and guanidine hydrochloride, to produce an extract;
- (c) passing said extract through a first ultra-filtration membrane having a molecular cutoff of from 20,000 to 50,000 daltons, to obtain a filtrate;
- (d) concentrating said filtrate by passing it through a second ultrafiltration membrane having a molecular cutoff of from 5,000 to 10,000, to produce a retentate;
- (e) subjecting said retentate to cation exchange chromatography at a pH of from about 5 to 6, using an elution buffer which comprises urea, and eluting a solution containing a peak of endotoxin binding activity;
- and
- (f) applying said solution containing said peak of endotoxin binding activity to a reverse phase column, and adding a buffer to said reverse phase column, to obtain a solution containing purified endotoxin binding protein.
A pharmaceutical composition effective for amelio-rating the biological effects of endotoxin in vivo when administered to a mammal, comprising a purified endotoxin-binding protein of the horseshoe crab and a pharmaceutically acceptable carrier.
A pharmaceutical composition effective for amelio-rating the biological effects of endotoxin in vivo when administered to a mammal, comprising a purified endotoxin-binding protein preparation and a pharmaceutically acceptable carrier, wherein said protein preparation is obtained according to the following process:
- (a) subjecting amebocytes obtained from a horse-shoe crab to hypotonic shock to lyse said amebocytes, and obtain cell debris from said lysed amebocytes;
- (b) extracting said cell debris with a solution containing a denaturant selected from the group consisting of urea and guanidine hydrochloride, to produce an extract;
- (c) passing said extract through a first ultra-filtration membrane having a molecular cutoff of from 20,000 to 50,000 daltons, to obtain a filtrate;
- (d) concentrating said filtrate by passing it through a second ultrafiltration membrane having a molecular cutoff of from 5,000 to 10,000, to produce a retentate;
- (e) subjecting said retentate to cation exchange chromatography at a pH of from about 5 to 6, using an elution buffer which comprises urea, and eluting a solution containing a peak of endotoxin binding activity;
- and
- (f) applying said solution containing said peak of endotoxin binding activity to a reverse phase column, and adding a buffer to said reverse phase column, to obtain a solution containing purified endotoxin binding protein.
A method for ameliorating the biological effects of endotoxin in vivo, which comprises administering to a mammal in need thereof an effective amount of an endotoxin binding protein having amino acid sequence SEQ. ID. NO. I.
A pharmaceutical composition for administration to a mammal to ameliorate the biological effects of endotoxin in vivo, which comprises an endotoxin binding protein having amino acid sequence SEQ. ID. NO. I in combination with a pharmaceutically acceptable carrier material.
A method for ameliorating the biological effects of endotoxin in vivo which comprises administering to a mammal in need thereof an effective amount of an endotoxin binding protein having an amino acid sequence corresponding at least to amino acid position 30 to 55 of SEQ. ID. NO. III and up to complete SEQ. ID. NO. III.
A pharmaceutical composition for administration to a mammal to ameliorate the biological effects of endotoxin in vivo, which comprises an endotoxin binding protein having an amino acid sequence corresponding at least to amino acid positions 30 to 55 of SEQ. ID. NO. III and up to complete amino SEQ. ID. NO. III, in combination with a pharmaceutically acceptable carrier material.
An endotoxin binding protein having amino acid sequence SEQ. ID. No. I free of the contaminating components naturally associated with the horseshoe crab.
An endotoxin binding protein having an amino acid sequence corresponding at least to amino acid positions 30 to 55 of SEQ. ID. No. III and up to complete amino SEQ. ID. NO. III free of the contaminating components naturally associated with the horseshoe crab.
A DNA molecule coding for an endotoxin binding protein having amino acid sequence SEQ. ID. NO. I, wherein said DNA molecule is free of the contaminating components naturally associated with the horseshoe crab.
A DNA molecule coding for an endotoxin binding protein having an amino acid sequence corresponding at least to amino acid positions 30 to 55 of SEQ. ID. NO. III and up to complete amino SEQ. ID. NO. III, wherein said DNA molecule is free of the contaminating components naturally associated with the horseshoe crab.
Dependencies are inferred from the claim text.