Polymer grafting by polysaccharide synthases

• US 20060183203A1
• Filed: 04/24/2006
• Published: 08/17/2006
• Est. Priority Date: 11/10/1999
• Status: Active Grant

• Current Assignee: Paul L. Deangelis
• Original Assignee: Paul L. Deangelis
• Inventors: DeAngelis, Paul L.
• Granted Patent: US 7,534,589 B2
• Time in Patent Office: Days
• US Class Current: 435/85
• CPC Class Codes: C08B 37/0069 Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• 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 recombinant, soluble hyaluronic acid synthase has an amino acid sequence as set forth in SEQ ID NO;

1; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the recombinant, soluble 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 recombinant, soluble hyaluronic acid synthase is isolated from a recombinant host cell capable of producing the recombinant, soluble hyaluronic acid synthase, and wherein the recombinant host cell lacks the ability to incorporate GlcUA or GlcNAc.

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 recombinant, soluble hyaluronic acid synthase has an amino acid sequence that is at least about 87% identical to SEQ ID NO;

1; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the recombinant, soluble 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. The method of claim 5, wherein the recombinant, soluble hyaluronic acid synthase is isolated from a recombinant host cell capable of producing the recombinant, soluble hyaluronic acid synthase, and wherein the recombinant host cell lacks the ability to incorporate GlcUA or GlcNAc.

9. 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 recombinant, soluble 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 a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO;

1; and

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

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

11. The method of claim 9, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

12. The method of claim 9, wherein the recombinant, soluble hyaluronic acid synthase is isolated from a recombinant host cell capable of producing the recombinant, soluble hyaluronic acid synthase, and wherein the recombinant host cell lacks the ability to incorporate GlcUA or GlcNAc.