Polymer grafting by polysaccharide synthases

• US 7,060,469 B2
• Filed: 07/16/2002
• Issued: 06/13/2006
• Est. Priority Date: 04/02/1998
• Status: Expired due to Fees

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul L.
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US10/197,153
• Publication Number:
  US 20050124046A1
• Time in Patent Office: 1,428 Days
• Field of Search: 435/97, 435/101, 435/194, 435/252.33, 536/23.2
• US Class Current: 435/101
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate synthase from Pasteurella multocida.

Claims

1. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, GalNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a recombinant, soluble hyaluronic acid synthase having an empty acceptor site and being capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing at 68°

C. In 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS, followed with washing in 0.2×

SSC/0.1% SDS at room temperature to a complement of the hyaluronic acid synthase nucleotide sequence as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

2. The method of claim 1, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

3. The method of claim 1, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

4. The method of claim 1, wherein the hyaluronic acid synthase is isolated from a source capable of producing the hyaluronic acid synthase, wherein the source capable of producing the hyaluronic acid synthase lacks the ablilty to incorporate GlcUA, GlcNAc, or GalNAc.

5. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, GalNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a recombinant, soluble hyaluronic acid synthase having an empty acceptor site and being capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

6. The method of claim 5, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

7. The method of claim 5, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

8. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, GalNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a recombinant, soluble hyaluronic acid synthase having an empty acceptor site and being capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing at 68°

C. in 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS, followed with washing in 0.2×

SSC/0.1% SDS at room temperature to a complement of the hyaluronic acid synthase nucleotide sequence as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

9. The method of claim 8, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

10. The method of claim 8, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

11. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, GalNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a recombinant, soluble hyaluronic acid synthase having an empty acceptor site and being capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ. ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

12. The method of claim 11, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

13. The method of claim 11, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.