Methods of producing testosteronan polymers using testosteronan synthase
View Patent ↗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 .
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.