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US 7,642,071 B2Granted
Methods of expressing gram-negative glycosaminoglycan synthase genes in gram-positive hosts
AssigneeThe Board of Regents of the University of Oklahoma
InventorPaul L. DeAngelis
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Abstract
The present invention relates to a Gram-negative glycosaminoglycan gene and methods of making and using same. The present invention relates to recombinant Gram-positive host cells containing a Gram-negative glycosaminoglycan synthase gene, and methods of producing glycosaminoglycans using such recombinant host cells.
Claims19
A method for producing a chondroitin polymer in vivo, comprising the steps of:
- providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, wherein the chondroitin synthase is a single protein that is a dual-action transferase that catalyzes the polymerization of UDP-GIcUA and UDP-GaINAc to form chondroitin, and wherein the purified nucleic acid segment comprises at least one of:
- (A) the nucleic acid sequence of SEQ ID NO:1;
- (B) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2;
- (C) a nucleic acid sequence that is at least 80% identical to SEQ ID NO:1;
- and
- (D) a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to the entirety of SEQ ID NO:2;
- providing a Gram-positive host cell;
- placing the purified nucleic acid segment encoding the enzymatically active chondroitin synthase in the Gram-positive host cell, thereby providing a recombinant Gram-positive host cell having a purified nucleic acid segment encoding an enzymatically active chondroitin synthase therein;
- placing the recombinant Gram-positive host cell in a medium suitable for the expression of the enzymatically active chondroitin synthase, whereby a chondroitin polymer is produced;
- and
- isolating the chondroitin polymer.
8 more elements
A method for producing a chondroitin polymer in vivo, comprising the steps of:
- providing a purified nucleic acid segment encoding an enzymatically active chondroitin synthase, wherein the chondroitin synthase is a single protein that is a dual-action transferase that catalyzes the polymerization of UDP-GIcUA and UDP-GaINAc to form chondroitin, and wherein the purified nucleic acid segment comprises at least one of:
- (A) the nucleic acid sequence of SEQ ID NO:1;
- (B) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2;
- (C) a nucleic acid sequence that is at least 800/a identical to SEQ ID NO:1;
- and
- (D) a nucleic acid sequence that encodes an amino acid sequence that is at least 900/a identical to the entirety of SEQ ID NO:2;
- providing a Bacillus host cell, wherein the Bacillus host cell comprises nucleic acid segments encoding enzymes which produce UDP-GIcUA and UDP-GaINAc;
- placing the purified nucleic acid segment encoding the enzymatically active chondroitin synthase in the Bacillus host cell, thereby providing a recombinant Bacillus host cell having a purified nucleic acid segment encoding an enzymatically active chondroitin synthase therein;
- placing the recombinant Bacillus host cell in a medium suitable for the expression of the enzymatically active chondroitin synthase, whereby a chondroitin polymer is produced;
- and
- isolating the chondroitin polymer.
8 more elements
Dependencies are inferred from the claim text.