Methods of producing testosteronan polymers using testosteronan synthase

Patent metadata

Field	Value
Publication number	US-9695427-B2
Application number	US-201213981886-A
Country	US
Kind code	B2
Filing date	Jan 31, 2012
Priority date	Jan 31, 2011
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

Testosteronan, a heparosan analog having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n , is produced by testosteronan synthase, a single protein that is a dual-action catalyst that utilizes UDP-GlcUA and UDP-GlcNAc to synthesize a polysaccharide having the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n .

First claim

What is claimed is:

1. A method of producing a polymer comprising the structure [4-D-glucuronic acid-α1,4-D-N-acetylglucosamine-α1-] n ([-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n ), the method comprising the steps of:
culturing a recombinant host cell that produces the polypeptide of SEQ ID NO:1;
isolating the polypeptide of SEQ ID NO:1; and
contacting the polypeptide of SEQ ID NO:1 with at least one UDP-sugar and a functional acceptor, wherein said UDP-sugar is selected from the group consisting of UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetyl-glucosamine (UDP-GlcNAc), wherein the functional acceptor is elongated to produce a polymer comprising the structure [-4-D-GlcUA-α1,4-D-GlcNAc-α1-] n , and wherein the functional acceptor comprises at least two sugar units, wherein said sugar units are selected from the group consisting of uronic acid, a uronic acid analog which results from a substitution at C2 and/or C3 in uronic acid, hexosamine, and a hexosamine analog which results from a substitution at C2 and/or C6 in hexosamine.

2. The method of claim 1 , wherein the at least one UDP-sugar is provided in a stoichiometric ratio to the functional acceptor such that the polypeptide of SEQ ID NO:1 elongates the functional acceptor to provide a polysaccharide having a desired size distribution such that the polysaccharide is substantially monodisperse in size and has a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.

3. The method of claim 1 , wherein the uronic acid is selected from the group consisting of GlcUA, iduronic acid (IdoUA) and GalUA; the hexosamine is selected from the group consisting of GlcNAc, GalNAc, GlcN, and GalN; the uronic acid analog is selected from the group consisting of GlcNAcUA, GlcdiNAcUA, and 2-deoxy-2-fluoro-GlcUA; and the hexosamine analog is selected from the group consisting of GlcN, GlcNAcNAc, GlcN[TFA], GlcNBut, GlcNPro, and 6-F-6-deoxyGlcNAc.

4. The method of claim 1 , wherein the at least one UDP-sugar is radioactive or nuclear magnetic resonance-active.

5. The method of claim 1 , further comprising the step of providing one or more divalent metal ions, wherein the divalent metal ions are selected from the group consisting of manganese, magnesium, cobalt, and nickel ions, and wherein the method is carried out in a buffer having a pH from about 4 to about 9.

6. The method of claim 1 , wherein the functional acceptor further comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag, a radioactive therapeutic, an affinity tag, a detection probe, a medicant, a biologically active agent, a therapeutic agent, and combinations thereof.

7. The method of claim 1 , wherein the functional acceptor comprises at least one testosteronan oligosaccharide, testosteronan polysaccharide, testosteronan polymer, heparosan oligosaccharide, heparosan polysaccharide, heparosan polymer, GlcUA-based glycoside, or GlcUA-analog glycoside.

Assignees

• Deangelis Paul L
• Otto Nigel J
• Univ Oklahoma

Inventors

• Deangelis Paul L
• Otto Nigel J

Classifications

• C12Y101/01022

  UDP-glucose 6-dehydrogenase (1.1.1.22) · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12Y207/07009

  UTP-glucose-1-phosphate uridylyltransferase (2.7.7.9), i.e. UDP-glucose-pyrophosphorylase · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title