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Methods of producing hyaluronic acid and chimeric and hybrid glycosaminoglycan polymers

  • US 7,741,091 B2
  • Filed: 01/25/2007
  • Issued: 06/22/2010
  • Est. Priority Date: 04/02/1998
  • Status: Expired due to Fees
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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