Compositions of proteus vulgaris chondroitinase I and chondroitinase II
First Claim
1. A surgical composition consisting essentially of a mixture of Proteus vulgaris chondroitinase I Proteus vulgaris and chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
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Accused Products
Abstract
Chondroitinase II, a protein having an isoelectric point of approximately 8.4-8.45 and an apparent molecular mass of 112 kDa when electrophoresed in a 4 to 20% gradient acrylamide gel in 25 mM Tris/192 mM glycine buffer at pH 8.5 in the presence of about 0.1% (w/v) SDS, has been isolated and purified. A process for the copurification by affinity chromatography of the chondroitinase I and chondroitinase II proteins produced by Proteus vulgaris is also provided. The proteins can be further purified by metal chelating chromatography. Therapeutic or surgical compositions of isolated chondroitinases I and II or the copurified mixture of chondroitinase I and II are also disclosed. These compositions are used in a method for selectively and completely disinserting the vitreous body from the neural retina of an eye.
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Citations
43 Claims
- 1. A surgical composition consisting essentially of a mixture of Proteus vulgaris chondroitinase I Proteus vulgaris and chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
- 7. A surgical composition consisting essentially of a disinsertion effective amount of a mixture of Proteus vulgaris chondroitinase I and Proteus vulgaris chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
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9. A surgical composition consistently essentially of a mixture of Proteus vulgaris chondroitinase I and Proteus vulgaris chondroitinase II, wherein said chondroitinase I and chondroitinase II are prepared by a copurification process comprising:
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(a) preparing a clarified homogenate of induced P. vulgaris, said homogenate having a pH of 5.8 to 7.4; (b) loading said homogenate onto a negatively charged cation exchange resin chromatographic support so that any positively charged proteins comprising chondroitinase I and chondroitinase II in said homogenate form a non-covalent bond with said negatively charged support; (c) affinity-eluting, in pools, said chondroitinase proteins from said support with an aqueous solution of chondroitin sulfate at pH of 7.0-9.5; (d) loading said affinity etched protein pools onto an anion exchange resin chromatographic support to yield an unbound eluate; and (e) recovering said chondroitinase I and chondroitinase II proteins in said unbound elute, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II. - View Dependent Claims (10, 11, 12, 13, 14, 15)
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- 16. A surgical composition comprising a solution consisting essentially of a mixture of Proteus vulgaris chondroitinase I and Proteus vulgaris chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
- 18. A surgical composition comprising copurified Proteus vulgaris chondroitinase I and chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
- 25. A surgical composition comprising a disinsertion effective amount of copurified Proteus vulgaris chondroitinase I and chondroitinase II, wherein said composition is suitable for use in humans, and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II.
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33. A therapeutic composition of matter for selectively and completely disinserting the ocular vitreous body from the neural retina of the human eye, by administering to the eye of a human an effective amount of said composition wherein said composition comprises an isolated and purified chondroitinase II protein having an amino acid sequence corresponding to SEQ ID NO:
- 2 and an isoelectric point of approximately 8.4-8.45, and an isolated and purified chondroitinase I protein having an amino acid sequence corresponding to SEQ ID NO;
1, and wherein said chondroitinase I and chondroitinase II are prepared by a copurification process comprising;(a) preparing a clarified homogenate of induced P. vulgaris, said homogenate having a pH of 5.8 to 7.4%; (b) loading said homogenate onto a negatively charged cation exchange resin chromatographic support so that any positively charged proteins comprising chondroitinase I and chondroitinase II in said homogenate form a non-covalent bond with said negatively charged support; (c) affinity-eluting, in pools, said chondroitinase proteins from said support with an aqueous solution of chondroitin sulfate at pH of 7.0-9.5; (d) loading said affinity eluted protein pools onto an anion exchange resin chromatographic support to yield an unbound eluate; and (e) recovering said chondroitinase I and chondroitinase II proteins in said unbound elute and wherein said composition is essentially free of proteolytic degradation products of said chondroitinase I and chondroitinase II. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
- 2 and an isoelectric point of approximately 8.4-8.45, and an isolated and purified chondroitinase I protein having an amino acid sequence corresponding to SEQ ID NO;
Specification