DNA encoding hyaluronan synthase from Pasteurella multocida and methods of use
First Claim
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1. A method for producing hyaluronic acid, comprising the steps of:
- introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurellainto a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the nucleic acid segment is selected from the group consisting of;
(a) the nucleic acid sequence as set forth in SEQ ID NO;
2;
(b) a nucleic acid segment encoding SEQ ID NO;
1; and
(c) a nucleic acid segment encoding SEQ ID NO;
17;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid.
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Abstract
The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Pasteurella multocida hyaluronate synthase (PmHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product.
37 Citations
15 Claims
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1. A method for producing hyaluronic acid, comprising the steps of:
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introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurellainto a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the nucleic acid segment is selected from the group consisting of;
(a) the nucleic acid sequence as set forth in SEQ ID NO;
2;
(b) a nucleic acid segment encoding SEQ ID NO;
1; and
(c) a nucleic acid segment encoding SEQ ID NO;
17;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid. - View Dependent Claims (2, 3)
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4. A method for producing hyaluronic acid, comprising the steps of:
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introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein a complement of the nucleic acid segment is capable of hybridizing to the nucleic acid sequence of SEQ ID NO;
2 under standard hybridization conditions comprising 1.2-1.8×
HPB at 40-50°
C.;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid. - View Dependent Claims (5, 6)
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7. A method for producing hyaluronic acid, comprising the steps of:
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introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the nucleic acid segment is at least 85% identical to SEQ ID NO;
2;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid. - View Dependent Claims (8, 9)
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10. A method for producing hyaluronic acid, comprising the steps of:
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introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the enzymatically active hyaluronan synthase encoded by the purified nucleic acid segment is at least 89% identical to SEQ ID NO;
1;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid. - View Dependent Claims (11, 12)
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13. A method for producing hyaluronic acid, comprising the steps of:
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introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the hyaluronan synthase has SEQ ID NO;
18 and at least two DSD motifs therein, and wherein a complement of the nucleic acid segment is capable of hybridizing to the nucleic acid sequence of SEQ ID NO;
2 under standard hybridization conditions comprising 1.2-1.8×
HPB at 40-50°
C.;
growing the host organism in a medium to secrete hyaluronic acid; and
recovering the secreted hyaluronic acid. - View Dependent Claims (14, 15)
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Specification