US2004224393A1PendingUtilityA1
Modified human granulocyte-colony stimulating factor and process for producing same
Priority: Jul 8, 1999Filed: Jan 9, 2002Published: Nov 11, 2004
Est. expiryJul 8, 2019(expired)· nominal 20-yr term from priority
C07K 2319/75A61P 35/02C07K 2319/034C12N 15/70C07K 14/535C12N 15/625C12N 15/11
54
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Claims
Abstract
A modified human granulocyte-colony stimulating factor(hG-CSF) is produced by culturing a microorganism transformed with an expression vector comprising a gene encoding a modified hG-CSF to produce and secrete the modified hG-CSF to periplasm, said modified hG-CSF being obtained by replacing at least one of the 1st, 2nd, 3rd and 17th amino acids of wild-type hG-CSF(SEQ ID NO: 2) with other amino acid
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A modified human granulocyte-colony stimulating factor(hG-CSF) which is characterized in that at least one of the 1st, 2nd, 3rd and 17th amino acids of wild-type hG-CSF(SEQ ID NO: 2) is replaced by other amino acid(s).
2 . The modified hG-CSF of claim 1 whose amino acid sequence is the same as that of wild-type hG-CSF, except that
(a) the 1st amino acid is Ser;
(b) the 1st amino acid is Ser and the 17th amino acid is X;
(c) the 2nd amino acid is Met and the 3rd amino acid is Val;
(d) the 2nd amino acid is Met, the 3rd amino acid is Val and the 17th amino acid is X; or
(f) the 17th amino acid is X,
wherein X is an amino acid which is not charged at neutral pH.
3 . The modified hG-CSF of claim 2 , wherein X is Ser, Thr, Ala or Gly.
4 . The modified hG-CSF of claim 3 , wherein X is Ser.
5 . A DNA encoding the modified hG-CSF of any one of claims 1 to 4 .
6 . The DNA of claim 5 , wherein the 1st to the 60th nucleotide sequence of the modified hG-CSF DNA corresponds to one selected from the group consisting of SEQ ID NOS: 55, 57, 59, 61, 63, 65, 67 and 69.
7 . An expression vector comprising the DNA of claim 5 .
8 . The expression vector of claim 7 , which further comprises a polynucleotide encoding a signal peptide attached at the 5′-end of the DNA encoding the modified hG-CSF.
9 . The expression vector of claim 8 , wherein the signal peptide is E. coli thermoresistant enterotoxin II signal peptide or modified E. coli thermoresistant enterotoxin II signal peptide.
10 . The expression vector of claim 9 , wherein the E. coli thermoresistant enterotoxin II signal peptide has the amino acid sequence of SEQ ID NO: 53.
11 . The expression vector of claim 9 , wherein the modified E. coli thermoresistant enterotoxin II signal peptide has the amino acid sequence of SEQ ID NO: 54.
12 . The expression vector of claim 9 , which further comprises a modified E. Coli enterotoxin II Shine-Dalgano sequence having the nucleotide sequence of SEQ ID NO: 71.
13 . The expression vector of claim 8 , wherein the signal peptide is E. coli beta lactamase signal peptide or modified E. coli beta lactamase signal peptide.
14 . The expression vector of claim 13 , wherein the E. coli beta lactamase signal peptide has the amino acid sequence of SEQ ID NO: 24.
15 . The expression vector of claim 8 , wherein the signal peptide is E. coli Gene III signal peptide or modified E. coli Gene III signal peptide.
16 . The expression vector of claim 15 , wherein the E. coli Gene III signal peptide has the amino acid sequence of SEQ ID NO: 42.
17 . The expression vector of claim 7 or 8 , which is pT14SS1SG, pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
18 . A microorganism transformed with the expression vector according to claim 7 or 8 .
19 . The microorganism of claim 18 , which is a transformed E. coli.
20 . The microorganism of claim 19 , wherein the transformed E. coli is E. coli BL21(DE3)/pT14SS1SG(HM 10310), E. coli BL21(DE3)/pT14SS1S17SEG(HM 10311, KCCM-10154), E. coli BL21(DE3)/pTO1SG(HM 10409), E. coli BL21(DE3)/pTO1S17SG(HM 10410, KCCM-10151), E. coli BL21(DE3)/pTO17SG(HM 10411, KCCM-10152), E. coli BL21 (DE3)/pTO17TG(HM 10413), E. coli BL21 (DE3)/pTO17AG(HM 10414), E. coli BL21(DE3)/pTO17GG(HM 10415), E. coli BL21(DE3)/pBAD2M3VG(HM 10510, KCCM-10153), E. coli BL21(DE3)/pBAD17SG(HM 10511) or E. coli BL21(DE3)/pBAD2M3V 17SG(HM 10512).
21 . A process for producing a modified hG-CSF in microorganism which comprises culturing the transformed microorganism of claim 18 to produce and secrete the modified hG-CSF to periplasm. E. coli thermoresistant enterotoxin II signal peptide or modified E. coli thermoresistant enterotoxin II signal peptide.
10 . The expression vector of claim 9 , wherein the E. coli thermoresistant enterotoxin II signal peptide has the amino acid sequence of SEQ ID NO: 53.
11 . The expression vector of claim 9 , wherein the modified E. coli thermoresistant enterotoxin II signal peptide has the amino acid sequence of SEQ ID NO: 54.
12 . The expression vector of claim 9 , which further comprises a modified E. coli enterotoxin II Shine-Dalgano sequence having the nucleotide sequence of SEQ ID NO: 71.
13 . The expression vector of claim 8 , wherein the signal peptide is E. coli beta lactamase signal peptide or modified E. coli beta lactamase signal peptide.
14 . The expression vector of claim 13 , wherein the E. coli beta lactamase signal peptide has the amino acid sequence of SEQ ID NO: 24.
15 . The expression vector of claim 8 , wherein the signal peptide is E. coli Gene III signal peptide or modified E. coli Gene III signal peptide.
16 . The expression vector of claim 15 , wherein the E. coli Gene III signal peptide has the amino acid sequence of SEQ ID NO: 42.
17 . The expression vector of claim 7 or 8 , which is pT14SS1SG, pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
18 . A microorganism transformed with the expression vector according to claim 7 or 8 .
19 . The microorganism of claim 18 , which is a transformed E. coli .Join the waitlist — get patent alerts
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