Methods of producing hyaluronic acid and chimeric and hybrid glycosaminoglycan polymers
First Claim
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1. A method for enzymatically producing a hyaluronic acid polymer in vitro, wherein the hyaluronic acid polymer is composed of less than about 150 sugars, comprising the steps of:
- providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine;
providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is selected from the group consisting of;
(a) a hyaluronic acid synthase having the amino acid sequence as set forth in SEQ ID NO;
2;
(b) a hyaluronic acid synthase encoded by the nucleotide sequence as set forth in SEQ ID NO;
1;
(c) a truncated form of (a) encoded by the nucleotide sequence as set forth in any of SEQ ID NOS;
20, 27-30 and 71;
(d) a truncated form of (a) having the amino acid sequence as set forth in SEQ ID NO;
9;
(e) a mutated form of (a) encoded by the nucleotide sequence as set forth in SEQ ID NO;
42; and
(f) a hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO;
1 under hybridization conditions selected from the group consisting of;
(i) 5×
SSC/5×
Denhardt'"'"'s solution/1.0% SDS at 68°
C., followed with washing in 0.2×
SSC/0.1% SDS at room temperature;
(ii) 1.8×
HPB at about 30°
C. to about 45°
C. followed by washing in 0.2-0.5×
HPB at about 45°
C.; and
(iii) 5×
SSC/5×
Denhardt'"'"'s solution/1% SDS at 68°
C., followed with washing in 3×
SSC at 42°
C.; and
providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor to provide a hyaluronic acid polymer wherein the hyaluronic acid polymer is composed of less than about 150 sugars.
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Abstract
The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate or chondroitin or heparin/heparosan synthases from Pasteurella multocida, in order to create a variety of glycosaminoglycan oligosaccharides having a natural or chimeric or hybrid sugar structure.
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Citations
10 Claims
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1. A method for enzymatically producing a hyaluronic acid polymer in vitro, wherein the hyaluronic acid polymer is composed of less than about 150 sugars, comprising the steps of:
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providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine; providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is selected from the group consisting of; (a) a hyaluronic acid synthase having the amino acid sequence as set forth in SEQ ID NO;
2;(b) a hyaluronic acid synthase encoded by the nucleotide sequence as set forth in SEQ ID NO;
1;(c) a truncated form of (a) encoded by the nucleotide sequence as set forth in any of SEQ ID NOS;
20, 27-30 and 71;(d) a truncated form of (a) having the amino acid sequence as set forth in SEQ ID NO;
9;(e) a mutated form of (a) encoded by the nucleotide sequence as set forth in SEQ ID NO;
42; and(f) a hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO;
1 under hybridization conditions selected from the group consisting of;(i) 5×
SSC/5×
Denhardt'"'"'s solution/1.0% SDS at 68°
C., followed with washing in 0.2×
SSC/0.1% SDS at room temperature;(ii) 1.8×
HPB at about 30°
C. to about 45°
C. followed by washing in 0.2-0.5×
HPB at about 45°
C.; and(iii) 5×
SSC/5×
Denhardt'"'"'s solution/1% SDS at 68°
C., followed with washing in 3×
SSC at 42°
C.; andproviding UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor to provide a hyaluronic acid polymer wherein the hyaluronic acid polymer is composed of less than about 150 sugars. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for producing a chimeric or hybrid glycosaminoglycan having a non-natural structure, wherein the chimeric or hybrid glycosaminoglycan comprises at least one sugar not found in a single naturally occurring glycosaminoglycan, the method comprising the steps of:
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providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine, and wherein at least one of the at least two sugar units is a sugar unit other than GlcUA and GlcNAc; providing at least one hyaluronic acid synthase selected from the group consisting of; (a) a hyaluronic acid synthase having the amino acid sequence as set forth in SEQ ID NO;
2;(b) a hyaluronic acid synthase encoded by the nucleotide sequence as set forth in SEQ ID NO;
1;(c) a truncated form of (a) encoded by the nucleotide sequence as set forth in any of SEQ ID NOS;
20, 27-30 and 71;(d) a truncated form of (a) having the amino acid sequence as set forth in SEQ ID NO;
9;(e) a mutated form of (a) encoded by the nucleotide sequence as set forth in SEQ ID NO;
42;(f) a hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NQ;
1 under hybridization conditions selected from the group consisting of;(i) 5×
SSC/5×
Denhardt'"'"'s solution/1.0% SDS at 68°
C., followed with washing in 0.2×
SSC/0.1% SDS at room temperature;(ii) 1.8×
HPB at about 30°
C. to about 45°
C. followed by washing in 0.2-0.5×
HPB at about 45°
C.; and(iii) 5×
SSC/5×
Denhardt'"'"'s solution/1% SDS at 68°
C., followed with washing in 3×
SSC at 42°
C.; and(g) at least one modified hyaluronic acid synthase, wherein the at least one modified hyaluronic acid synthase is a single action glycosyltransferase capable of adding only one of GlcUA or GlcNAc to a functional acceptor, and wherein the at least one modified hyaluronic acid synthase is selected from the group consisting of; (i) a modified hyaluronic acid synthase having the nucleotide sequence as set forth in any of SEQ ID NQS;
10-12, 16-19, 31-41, and 43-50; and(ii) a modified hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO;
1 under the hybridization conditions of (f);providing at least one of UDP-GlcUA and UDP-GlcNAc such that the at least one hyaluronic acid synthase elongates the functional acceptor in a manner so as to provide a chimeric or hybrid glycosaminoglycan having a non-natural structure whereby the chimeric or hybrid glycosaminoglycan comprises at least one sugar not found in a naturally occurring hyaluronic acid polymer.
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Specification