ASPART PROINSULIN COMPOSITIONS AND METHODS OF PRODUCING ASPART INSULIN ANALOGS THEREFROM
Aspart modified proinsulin sequences that have a modified C-peptide amino acid and/or nucleic acid modification for producing aspart insulin analogs are provided. Highly efficient processes for preparing the aspart insulin analogs and improved preparations containing the aspart insulin analogs prepared according to the methods described herein are also provided.
1 . A composition comprising a modified aspart proinsulin sequence having the formula
R 1 —(B 1 —B 26 )—B 27 —B 28 —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 26 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 27 is Thr, Asp, or Glu;
B 28 is Asp, Glu, or Pro;
B 29 is Lys, or Pro;
B 30 is Ala, Thr, or is absent;
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 Asn, 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 RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO: 8).
6 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is FVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGGGPGAGSLQPLAL EGSLQARGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 15).
7 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGG G PGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 16).
8 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGG G PGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCNRHHHHHH (SEQ ID NO: 18).
9 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGG G PGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCNKHHHHHH (SEQ ID NO: 19).
10 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is MRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGGGPGAGSLQP L ALEGSLQARGIVEQCCTSICSLYQLENYCNRHHHHHH (SEQ ID NO: 20).
11 . The composition of claim 1 , wherein the modified aspart proinsulin sequence is MRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRREAEDLQVGQVELGGGPGAGSLQP L ALEGSLQARGIVEQCCTSICSLYQLENYCNKHHHHHH (SEQ ID NO: 21).
12 . An expression vector comprising the nucleic acid sequence of claim 1 .
13 . The expression vector of claim 12 , wherein the expression vector is His Tagged Aspart proinsulin pTrcHis2A(Kan).
14 . A microorganism transformed with the vector of claim 13 .
15 . The microorganism of claim 14 , further defined as an E. coli transformed with plasmid His Tagged Aspart proinsulin pTrcHis2A(Kan).
16 . A process for producing aspart insulin analogs comprising the steps of
(a) culturing under conditions suitable for expression E. coli cells having a modified proinsulin sequence of a formula:
R 1 —(B 1 —B 26 )—B 27 —B 28 —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 26 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 27 is Thr, Asp, or Glu;
B 28 is Asp, Glu, or Pro;
B 29 is Lys, or Pro;
B 30 is Ala, Thr, or is absent;
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 Asn, 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, to provide cultured E. coli cells;
(b) disrupting said cultured E. coli cells to provide a composition comprising inclusion bodies containing the modified aspart proinsulin sequence;
(c) solubilizing said composition of inclusion bodies; and
(d) recovering the aspart insulin analogs from said solubilized composition.
17 . The process of claim 16 , wherein the step of recovering the aspart insulin analogs further comprises:
(e) folding said modified aspart proinsulin sequence to provide an aspart proinsulin derivative peptide;
(f) purifying said aspart proinsulin derivative peptide using metal affinity chromatography;
(g) enzymatically cleaving aspart proinsulin derivative peptide to remove a connecting peptide and provide an intermediate solution comprising aspart insulin analog; and
(h) purifying said intermediate solution using chromatography column(s) to yield the aspart insulin analog.
18 . The process of claim 16 , wherein the solubilization of said composition of inclusion bodies further comprises adjusting the pH to at least 10.5.
19 . The process of claim 16 , wherein the solubilization of said composition of inclusion bodies comprises by adjusting the pH to 11.8 to 12.
20 . The process of claim 16 , wherein the solubilization of said composition of inclusion bodies includes one or more reducing agents selected from the group consisting of 2 imercaptoethanol, L-cysteine hydrochloride monohydrate, dithiothreitol, dithierythritol, and mixtures thereof.
21 . The process of claim 16 , wherein the solubilization of said composition of inclusion bodies includes the use one or more chaotropic agents selected from the group consisting of urea, thiourea, lithium perchlorate or guanidine hydrochloride and mixtures thereof.
22 . A process for producing aspart insulin analogs comprising the steps of:
(a) providing an E. coli transformed to include a modified aspart proinsulin sequence having a Formula I sequence under conditions suitable for expression of said sequence, Formula I as follows:
R 1 —(B 1 —B 26 )—B 27 —B 28 —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 26 ) and (A 1 -A 20 ) comprise amino acid sequences of native human insulin;
B 27 is Thr, Asp, or Glu;
B 28 is Asp, Glu, or Pro;
B 29 is Lys, or Pro;
B 30 is Ala, Thr, or is absent;
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 Asn, 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 modified aspart proinsulin collected from said culture to provide a proinsulin derivative peptide;
(c) purifying said aspart proinsulin derivative peptide using metal affinity chromatography;
(d) enzymatically cleaving said proinsulin derivative peptide to remove a connecting peptide and provide an intermediate solution comprising aspart insulin analog; and
(e) purifying said intermediate solution using chromatography column(s) to yield the aspart insulin analog.
23 . The process of claim 22 , wherein the step of purifying further comprises eluting the aspart insulin analog using a buffer.