GLARGINE PROINSULIN AND METHODS OF PRODUCING GLARGINE INSULIN ANALOGS THEREFROM
Glargine proinsulin sconstructs that have a modified C-peptide amino acid and/or nucleic acid sequence for producing glargine insulin analogs are provided. Highly efficient processes for preparing the glargine insulin analogs and improved preparations containing the glargine insulin analogs prepared according to the methods described herein are also provided.
1 . A composition comprising a proinsulin sequence having the formula
R 1 —(B 1 -B 29 )—B 30 —R 2 —R 3 —X—R 4 —R 5 -(A 1 -A 20 )-A 21 -R 6 Formula I
wherein
R 1 is a tag sequence containing one or more amino acids or R 1 is absent with an Arg or Lys present prior to the start of the B chain;
(B 1 -B 29 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 30 is Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, or Trp;
R 2 , R 3 and R 5 are Arg;
R 4 is any amino acid other than Gly, Lys or Arg or is absent;
X is a sequence comprises one or more amino acids or is absent, provided that X does not comprise a C-terminal Gly, Lys, or Arg when R 4 is absent;
A 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asp, or Glu; and
R 6 is a tag sequence containing one or more amino acids or R 6 is absent.
2 . The composition of claim 1 , wherein R 1 and/or R 6 is present and R 1 is tag sequence of one or more amino acids with a C-terminal Arg or Lys and/or R 6 tag sequence of one or more amino acids with a N-terminal Arg or Lys.
3 . The composition of claim 1 , wherein R 4 is Ala.
4 . The composition of claim 1 , wherein the modified proinsulin sequence comprises a connecting peptide sequence of a sequence having the formula
R 2 —R 3 —X—R 4 —R 5 Formula II
wherein
R 2 , R 3 , R 4 , R 5 , and X are defined in claim 1 .
5 . The composition of claim 4 , wherein the connecting peptide sequence is
(SEQ ID NO: 8)
RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR.
6 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 15)
FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPG
AGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCG.
7 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 17)
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGF
FYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQARGIVEQCC
TSICSLYQLENYCG.
8 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 16)
MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQ
VGQVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCG.
9 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 18)
MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQ
VGQVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCG
RHHHHHH.
10 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 19)
MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQ
VGQVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCG
KHHHHHH.
11 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 20)
MRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELG
GGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCGRHHHHH
H.
12 . The composition of claim 1 , wherein the modified proinsulin sequence is
(SEQ ID NO: 21)
MRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELG
GGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCGKHHHHHH.
13 . An expression vector comprising the nucleic acid sequence of claim 1 .
14 . The expression vector of claim 13 , wherein the expression vector is His Tagged Glargine proinsulin pTrcHis2A(Kan).
15 . A microorganism transformed with the vector of claim 14 .
16 . The microorganism of claim 15 , further defined as an E. coli transformed with plasmid His Tagged Glargine proinsulin pTrcHis2A(Kan).
17 . A process for producing glargine insulin analogs comprising the steps of:
(a) culturing E. coli cells under conditions suitable for expression of a modified proinsulin sequence having the formula
R 1 —(B 1 -B 29 )—B 30 —R 2 —R 3 —X—R 4 —R 5 -(A 1 -A 20 )-A 21 -R 6 Formula I
wherein
R 1 is a tag sequence containing one or more amino acids or R 1 is absent with an Arg or Lys present prior to the start of the B chain;
(B 1 -B 29 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 30 is Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, or Trp;
R 2 , R 3 and R 5 are Arg;
R 4 is any amino acid other than Gly, Lys or Arg or is absent;
X is a sequence comprises one or more amino acids or is absent, provided that X does not comprise a C-terminal Gly, Lys, or Arg when R 4 is absent;
A 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asp, or Glu; and
R 6 is a tag sequence containing one or more amino acids or R 6 is absent;
(b) disrupting said cultured E. coli cells to provide a composition comprising inclusion bodies containing the modified proinsulin sequence;
(c) solubilizing said composition of inclusion bodies; and
(d) recovering the glargine insulin analogs from said solubilized composition.
18 . The process of claim 17 , wherein the step of recovering the glargine insulin analogs further comprises:
(e) folding said modified proinsulin sequence to provide a proinsulin derivative peptide;
(f) purifying said proinsulin derivative peptide in a metal affinity chromatography column;
(g) protecting a Lys amino acid residue of the proinsulin derivative peptide with one or more protecting compounds;
(h) enzymatically cleaving said blocked proinsulin derivative peptide to remove a connecting peptide and provide an intermediate solution comprising glargine insulin analog; and
(i) purifying said intermediate solution using chromatography column(s) to yield the glargine insulin analog.
19 . The process of claim 18 , wherein the one or more protecting compounds comprise citriconic anhydride.
20 . The process of claim 17 , wherein the solubilization of said composition of inclusion bodies use one or more chaotropic agents selected from the group consisting of urea, thiourea, lithium perchlorate or guanidine hydrochloride and mixtures thereof.
21 . A process for producing glargine insulin analogs comprising the steps of:
(a) providing a modified proinsulin sequence having the formula
R 1 —(B 1 -B 29 )—B 30 —R 2 —R 3 —X—R 4 —R 5 -(A 1 -A 20 )-A 21 -R 6 Formula I
wherein
R 1 is a tag sequence containing one or more amino acids or R 1 is absent with an Arg or Lys present prior to the start of the B chain;
(B 1 -B 29 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 30 is Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, or Trp;
R 2 , R 3 and R 5 are Arg;
R 4 is any amino acid other than Gly, Lys or Arg or is absent;
X is a sequence comprises one or more amino acids or is absent, provided that X does not comprise a C-terminal Gly, Lys, or Arg when R 4 is absent;
A 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Tyr, Asp, or Glu; and
R 6 is a tag sequence containing one or more amino acids or R 6 is absent;
(b) folding said modified proinsulin sequence to provide a proinsulin derivative peptide;
(c) purifying said proinsulin derivative peptide in a metal affinity chromatography column;
(d) enzymatically cleaving said proinsulin derivative peptide to remove a connecting peptide and provide an intermediate solution comprising glargine insulin analog; and
(e) purifying said intermediate solution using chromatography column(s) to yield the glargine insulin analog.
22 . The process of claim 21 , further comprising
(f) protecting a lys amino acid residue of the proinsulin derivative peptide with one or more protecting compounds, prior to enzymatic cleavage.
23 . The process of claim 22 , wherein the one or more protecting compounds comprise citriconic anhydride.