Hyaluronan synthase gene and uses thereof
View Patent ↗The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active hyaluronate synthase (HAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.
1. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes at least two amino acid motifs selected from the group consisting of:
(i)
(SEQ ID NO:11)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)
(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(SEQ ID NO:12)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(SEQ ID NO:13)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
XGDDR(H/R/C)LTN;
and
(iv)
(SEQ ID NO:14)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.
2. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes at least three amino acid motifs selected from the group consisting of:
(i)
(SEQ ID NO:11)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)
(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(SEQ ID NO:12)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(SEQ ID NO:13)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
XGDDR(H/R/C)LTN;
and
(iv)
(SEQ ID NO:14)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.
3. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes the amino acid motifs:
(i)
(SEQ ID NO:11)
GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3–7
(A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)
(V/C/I)DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE;
(ii)
(SEQ ID NO:12)
(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)
(V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V)
(I/V/C)(S/G)GPL(G/S)XY(K/R);
(iii)
(SEQ ID NO:13)
(D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T)
XGDDR(H/R/C)LTN;
and
(iv)
(SEQ ID NO:14)
(E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N)
RW(S/T/N)KS(Y/W/F)(F/C)RE,
with X being selected from the group consisting of any amino acid; and
wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.
4. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes at least two amino acid motifs selected from the group consisting of:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID NO:17)
and
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.
5. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes at least three amino acid motifs selected from the group consisting of:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID NO:17)
and
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.
6. A recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase includes the amino acid motifs:
(i)
GKR(X) 6 F(X) 11–15 DSDT(X) 8 E;
(SEQ ID NO:15)
(ii)
RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y;
(SEQ ID NO:16)
(iii)
QXFXG(X) 5 GDDRXLTN;
(SEQ ID NO:17)
and
(iv)
P(X) 8 QQXRWXKS(X) 2 RE,
(SEQ ID NO:18)
with X being selected from the group consisting of any amino acid; and
wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.