Hyaluronate synthase gene and uses thereof
AssigneeThe Board of Regents of the University of Oklahoma
InventorsPaul H. Weigel, Paul L. DeAngelis, John Papaconstantinou
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Abstract
Disclosed are DNA sequences encoding hyaluronic acid synthase that are employed to construct recombinant cells useful in the production of hyaluronata synthase and hyaluronic acid (HA). In preferred aspects, chromosomal DNA encoding the HA synthase gene, hasA, was cloned from a Streptococcus pyoganas genomic library. These vectors were used to transform host cells such as E. coli and acapsular Streptococci to produce hyaluronic acid. Resultant transformants were screened to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies were selected and employed in the production of hyaluronic acid.
Claims28
A purified nucleic acid segment encoding HA synthase, wherein the segment:
- (a) encodes the HA synthase of SEQ ID NO:2;
- or
- (b) is capable of hybridizing to the complement of nucleotides 1–1449 of the nucleotide sequence of SEQ ID NO:1 under standard hybridization conditions selected from the group consisting of:
- (i) a temperature of about 37° C. in a hybridization mixture comprising NaCl at a concentration in a range of from about 0.5 M to about 1.5 M, a divalent cation chelator at a concentration in a range of from about 0.05 mM to about 0.5 mM, and a buffering agent at a concentration in a range of from about 10 mM to about 100 mM and having a pH of about 7.2;
- and
- (ii) a temperature of about 65° C. in a hybridization mixture comprising NaCl at a concentration in a range of from about 0.3 M to about 0.6 M, a divalent cation chelator at a concentration in a range of from about 0.05 mM to about 0.5 mM, and a buffering agent at a concentration in a range of from about 10 mM to about 100 mM and having a H of about 7.2.
A nucleic acid segment which comprises at least a 14 nucleotide long contiguous stretch which corresponds to, or is complementary to, least 14 of nucleptodes 1–1449 of the nucleic acid sequence of SEQ ID NO:1, and is capable of hybridizing to said nucleic acid segment under standard hybridization conditions selected from the group consisting of:
- (i) a temperature of about 37° C. in a hybridization mixture comprising NaCl at a concentration in a range of from about 0.5 M to about 1.5 M, a divalent cation chelator at a concentration in a range of from about 0.05 mM to about 0.5 mM, and a buffering agent at a concentration in a range of from about 10 mM to about 100 mM and having a pH of about 7.2;
- and
- (ii) a temperature of about 65° C. in a hybridization mixture comprising NaCl at a concentration in a range of from about 0.3 M to about 0.6 M, a divalent cation chelator at a concentration in a range of from about 0.05 mM to about 0.5 mM, and a buffering agent at a concentration in a range of from about 10 mM to about 100 mM and having a H of about 7.2.
A purified composition comprising a polypeptide having an amino acid sequence in accordance with SEQ ID NO:2.
Dependencies are inferred from the claim text.