Lis-pro proinsulin compositions and methods of producing lis-pro insulin analogs therefrom
AssigneeHorizon Bank Na
InventorsRonald E. Zimmerman, David J. Stokell, Michael P. Akers
Life of this patent
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Abstract
Lis-Pro modified proinsulin sequences that have a modified C-peptide amino acid and/or nucleic acid modification are presented. Methods for producing Lis-Pro insulin analogs are also disclosed. Highly efficient processes for preparing the Lis-Pro insulin analogs and improved preparations containing the Lis-Pro insulin analogs prepared according to the methods described herein are also provided.
Claims18
A composition comprising a Lis-Pro proinsulin sequence having a Formula I:
- R1-(B1-B27)-B28-B29-B30-R2-R3-X-R4-R5-(A1-A21)-R6 Formula I
- wherein
- R1 is a tag sequence containing one or more amino acids or R1 is absent with an Arg or Lys present prior to the start of the B chain;
- (B1-B27) and (A1-A21) comprise amino acid sequences of native human insulin;
- B28 is any amino acid other than Pro;
- B29 is any amino acid other than Lys or Arg;
- B30 is Thr;
- R2, R3 and R5 are Arg;
- R4 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 is not EAEALQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO:
- 2) and X does not comprise a C-terminal Gly, Lys, or Arg when R4 is absent;
- and
- R6 is a tag sequence containing one or more amino acids or R6 is absent.
A process for producing Lis-Pro insulin analogs comprising the steps of:
- (a) culturing transformed E. coli comprising a sequence of Formula I under conditions suitable for expression of a modified proinsulin sequence of Formula I:
- R1-(B1-B27)-B28-B29-B30-R2-R3-X-R4-R5-(A1-A21)-R6 Formula I
- wherein
- R1 is a tag sequence containing one or more amino acids or R1 is absent with an Arg or Lys present prior to the start of the B chain;
- (B1-B27) and (A1-A21) comprise amino acid sequences of native human insulin;
- B28 is any amino acid other than Pro;
- B29 is any amino acid other than Lys or Arg;
- B30 is Thr;
- R2, R3 and R5 are Arg;
- R4 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 is not EAEALQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO:
- 2) and X does not comprise a C-terminal Gly, Lys, or Arg when R4 is absent;
- and
- R6 is a tag sequence containing one or more amino acids or R6 is absent,
- (b) disrupting said cultured E. coli cells to provide a composition comprising inclusion bodies containing the modified Lis-Pro proinsulin;
- (c) solubilizing said composition of inclusion bodies;
- and
- (d) recovering the Lis-Pro insulin analogs from said solubilized composition.
The process of claim 16, wherein the solubilization of said composition of inclusion bodies includes one or more chaotropic agents selected from the group consisting of urea, thiourea, lithium perchlorate or guanidine hydrochloride and mixtures thereof.
A process for producing Lis-Pro insulin analogs comprising the steps of:
- (a) recombinantly producing a modified Lis-Pro proinsulin sequence having Formula I:
- R1-(B1-B27)-B28-B29-B30-R2-R3-X-R4-R5-(A1-A21)-R6 Formula I
- wherein
- R1 is a tag sequence containing one or more amino acids or R1 is absent with an Arg or Lys present prior to the start of the B chain;
- (B1-B27) and (A1-A21) comprise amino acid sequences of native human insulin;
- B28 is any amino acid other than Pro;
- B29 is any amino acid other than Lys or Arg;
- B30 is Thr;
- R2, R3 and R5 are Arg;
- R4 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 is not EAEALQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO:
- 2) and X does not comprise a C-terminal Gly, Lys, or Arg when R4 is absent;
- and
- R6 is a tag sequence containing one or more amino acids or R6 is absent,
- (b) folding said modified Lis-Pro proinsulin to provide a Lis-Pro proinsulin derivative peptide;
- (c) purifying said Lis-Pro proinsulin derivative peptide using metal affinity chromatography;
- (d) enzymatically cleaving said Lis-Pro proinsulin derivative peptide to remove a connecting peptide and provide an intermediate solution comprising Lis-Pro insulin analog;
- and
- (e) purifying said intermediate solution in a chromatography column to yield the Lis-Pro insulin analog.
Dependencies are inferred from the claim text.