Recombinant IGF expression systems
View Patent ↗The present invention provides nucleic acid sequences, expression systems, host cells, polypeptides and methods of producing mature insulin-like growth factor (IGF) using a fusion construct which yields high levels of fusion proteins that are easily processed to provide IGF with a proper N-terminus.
1. An isolated nucleic acid sequence encoding a polypeptide comprising the following sequence:
MQIFVKTLTGK[X 1 ]LE[X 2 ]LFQ[IGF]
(SEQ ID NO: 23)
wherein X 1 is a polypeptide having a sequence from 0 to 30 amino acids; X 2 is either V or T; and wherein the C-terminus of said polypeptide is fused to IGF.
2. The nucleic acid of claim 1 , wherein X 1 is selected from the group consisting of (A) zero amino acids, (B) six amino acids of the sequence TITLEV (SEQ ID NO: 24) and (C) 30 amino acids of the sequence
TITLEVESSDTIDNVKSKIQDKEGIPPDQQ.
(SEQ ID NO: 25)
3. A protein expression system, comprising a protein expression vector encoding the protein
MQIFVKTLTGK[X 1 ]LE[X 2 ]LFQ[IGF]
(SEQ ID NO: 23)
wherein X 1 is a polypeptide having a sequence from 0 to 30 amino acids; X 2 is either V or T; and wherein the C-terminus of said polypeptide is fused to IGF.
4. The protein expression system of claim 3 , wherein X 1 is selected from the group consisting of (A) zero amino acids, (B) six amino acids of the sequence TITLEV (SEQ ID NO: 24) and (C) 30 amino acids of the sequence
TITLEVESSDTIDNVKSKIQDKEGIPPDQQ.
(SEQ ID NO: 25)
5. A protein expression system, comprising a host cell and a protein expression vector encoding the polypeptide
MQIFVKTLTGK[X 1 ]LE[X 2 ]LFQ[IGF]
(SEQ ID NO: 23)
wherein X 1 is a polypeptide having a sequence from 0 to 30 amino acids; X 2 is either V or T; and wherein the C-terminus of said polypeptide is fused to IGF.
6. The protein expression system of claim 5 , wherein X 1 is selected from the group consisting of (A) zero amino acids, (B) six amino acids of the sequence TITLEV (SEQ ID NO: 24) and (C) 30 amino acids of the sequence
TITLEVESSDTIDNVKSKIQDKEGIPPDQQ.
(SEQ ID NO: 25)
7. The protein expression system of claim 5 , wherein the host cell is prokaryotic.
8. The protein expression system of claim 5 , wherein the host cell is eukaryotic.
9. A method of expressing IGF comprising:
(A) transfecting a host cell with an expression vector encoding the polypeptide
MQIFVKTLTGK[X 1 ]LE[X 2 ]LFQ[IGF]
(SEQ ID NO: 23)
wherein X 1 is a polypeptide having a sequence from 0 to 30 amino acids; X 2 is either V or T; and wherein the C-terminus of said polypeptide is fused to IGF.
(B) culturing said host cell under conditions that permit expression of said polypeptide; and
(C) isolating the polypeptide.
10. The method of claim 9 , wherein X 1 is selected from the group consisting of (A) zero amino acids, (B) six amino acids of the sequence TITLEV (SEQ ID NO: 24) and (C) 30 amino acids of the sequence
TITLEVESSDTIDNVKSKIQDKEGIPPDQQ.
(SEQ ID NO: 25)
11. The method of claim 9 , wherein the host cell is prokaryotic.
12. The method of claim 9 , wherein the host cell is eukaryotic.
13. An isolated nucleic acid sequence encoding a polypeptide comprising the polypeptide of SEQ ID NO: 23 and IGF, wherein the C-terminus of SEQ ID NO: 23 is fused to IGF.