US 12,325,732 B2
Pro-insulin aspart structure and method for preparing insulin aspart
Chuangen Tang, Jiangsu (CN); Shangshu Pan, Jiangsu (CN); Xiaorui Liu, Jiangsu (CN); Cheng Li, Jiangsu (CN); Huaiyan Cui, Jiangsu (CN); Song Chen, Jiangsu (CN); Haoning Zhang, Jiangsu (CN); and Jeffrey Ding, Jiangsu (CN)
Assigned to AMPHASTAR NANJING PHARMACEUTICALS INC, Jiangsu (CN)
Appl. No. 17/275,821
Filed by AMPHASTAR NANJING PHARMACEUTICALS INC, Jiangsu (CN)
PCT Filed Sep. 12, 2018, PCT No. PCT/CN2018/105314
§ 371(c)(1), (2) Date Mar. 12, 2021,
PCT Pub. No. WO2020/051812, PCT Pub. Date Mar. 19, 2020.
Prior Publication US 2021/0332100 A1, Oct. 28, 2021
Int. Cl. C07K 14/62 (2006.01)
CPC C07K 14/62 (2013.01) 9 Claims
 
1. A novel short-proinsulin aspart sequence for preparing recombinant insulin aspart and analogs thereof, wherein the sequence has an amino acid sequence as shown in Formula I:
R-R1-(B1-B27)-B28-B29-B30-R2-(A1-A21),
wherein R-R1 is a leading peptide sequence, which meets the following conditions:
(a) R is one part of super oxide dismutase (SOD) homolog, which consists of 63 amino acids including active methionine; and two cysteine (C) residues are substituted by serine(S);
wherein, the sequence of R is SEQ ID NO: 1:
MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE
 
FGDNTAGSTSAGP;
(b) the leading peptide does not affect refolding of short-proinsulin aspart and can be cleaved;
(c) R1 is any one of Arg and Lys,
R2 is a C-peptide, which consists of any one from Arg or Lys;
B1-B27 denotes an amino acid sequence of B1 to B27 in B-chain of native human insulin;
A1-A21 denotes an A-chain of native human insulin;
B28 is Aspartic acid;
B29 is Lysine; and
B30 is Threonine;
the sequence of (B1-B27)-B28-B29-B30-R2-(A1-A21) is one of SEQ ID NO: 12 or SEQ ID NO: 13;
a sequence of SEQ ID NO: 12 is:
FVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN; and
a sequence of SEQ ID NO: 13 is:
FVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQCCTSICSLYQLENY
 
CN.