Modified human granulocyte-colony stimulating factor and process for producing same
View Patent ↗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.
1. A modified human granulocyte-colony stimulating factor(hG-CSF) which is different from a parent wild-type hG-CSF of the amino acid sequence of SEQ ID NO: 2 by having at least one replacements of amino acid residues at the positions 1st, 2nd, 3rd and 17th amino acids of the wild-type hG-CSF,
wherein the replaced amino acids in the modified hG-CSF are different from one present in the parent wild-type hG-CSF;
wherein the modified hG-CSF has no sugar chain;
wherein the modified hG-CSF has no terminal Met residue at the N-terminus thereof;
wherein when the 1st amino acid is replaced, the first amino acid in the modified hG-CSF is Ser; and
when the 17th amino acid is replaced, the 17th amino acid in the modified hG-CSF is an amino acid which is not charged at neutral pH.
2. The modified hG-CSF of claim 1 having an amino acid sequence which is the same as that of the parent 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 selected from the group consisting of Ser, Thr, Ala and Gly.
4. The modified hG-CSF of claim 3 , wherein X is Ser.
5. A DNA encoding the modified hG-CSF of claim 1 .
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 an E.coli thermoresistant enterotoxin II signal peptide or a 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 7 , which is pT14SS1SG, pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
12. A microorganism transformed with the expression vector according to claim 7 .
13. The microorganism of claim 12 , which is a transformed E.coli.
14. The microorganism of claim 13 , wherein the transformed E.coli is selected from the group consisting of 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 BL 21(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)/pBAD2M3V17SG(HM 10512).
15. A process for producing a modified hG-CSF in microorganism which comprises culturing the transformed microorganism of claim 12 to produce and secrete the modified hG-CSF to a periplasm.
16. A DNA encoding the modified hG-CSF of claim 2 .
17. A DNA encoding the modified hG-CSF of claim 3 .
18. A DNA encoding the modified hG-CSF of claim 4 .
19. The expression vector of claim 8 , which is pT14SS1SG, pT14SS1S17SEG, pTO1SG, pTO1S17SG, pTO17SG or pBAD2M3V17SG.
20. A microorganism transformed with the expression vector according to claim 8 .
21. The DNA of claim 6 , wherein the 1st to the 60th nucleotide sequence of the modified hG-CSF DNA corresponds to SEQ ID NO: 59.
22. The modified hG-CSF of claim 1 , which has an amino acid sequence of SEQ ID NO: 60.
23. 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.
24. The expression vector of claim 9 , which further comprises a modified E coli beta enterotoxin II Shine-Dalgano sequence having the amino acid sequence of SEQ ID NO: 71.
25. 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.
26. The expression vector of claim 25 , wherein the E.coli beta lactamase signal peptide has the amino acid sequence of SEQ ID NO: 24.
27. The expression vector of claim 8 , wherein the signal peptide is bacteriophage fd Gene III signal peptide.
28. The expression vector of claim 27 , wherein the bacteriophage fd Gene III signal peptide has the amino acid sequence of SEQ ID NO: 42.