Insulin production methods and pro-insulin constructs
AssigneeElona Biotechnologies
InventorsRonald E. ZIMMERMAN, David John STOKELL
Life of this patent
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Abstract
Novel pro-insulin having specific amino acid and/or nucleic acid modifications suitable for improved methods of insulin production are provided. Novel and highly efficient processes for preparing the pro-insulin preparations and preparations containing them are also disclosed. The novel pro-insulin preparations may be converted into human insulin useful in therapeutic preparations. Novel peptides of the C-peptide, and N terminus, including RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR, and MHHHHHHGGR respectively are provided, as well as the unique nucleic acid molecules encoding them.
Claims12
-8. (canceled)
A process for preparing a composition enriched for an insulin analog employing a modified human pro-insulin peptide comprising:
- (a) preparing an isolated nucleic acid sequence encoding native human pro-insulin;
- (b) modifying the isolated nucleic acid sequence by providing a molecular tag on the modified pro-insulin sequence so as to provide a histidine-tagged modified pro-insulin peptide;
- (c) converting a lysine residue to an alanine residue at position 64 in a C-peptide region of the pro-insulin sequence to provide a modified pro-insulin derivative gene sequence;
- (d) inserting the modified pro-insulin derivative gene sequence into a suitable vector to provide a vector comprising the modified pro-insulin derivative gene sequence;
- (e) transfecting a culture of competent cells comprising E. coli cells with the vector to provide transformed E. coli cells;
- (f) culturing the transformed E. coli cells under conditions suitable for expression of the modified pro-insulin derivative gene sequence;
- (g) disrupting the population of transformed E. coli cells to provide a composition comprising inclusion bodies containing the modified human pro-insulin;
- (h) solubilizing the composition to provide a composition comprising unfolded peptide;
- (i) refolding the unfolded peptide to provide refolded human pro-insulin derivative peptide;
- (j) passing the composition over a metal chelating column comprising nickel chelate to purify the composition and collecting a purified preparation of the refolded human pro-insulin derivative peptide;
- (k) transforming the collected purified preparation of refolded human pro-insulin derivative peptide into Arg and Di-Arg peptide species by tryptic digestion;
- (l) purifying the Arg and Di-Arg peptide species by passing the peptides through a reverse phase column;
- and
- (m) transforming the purified Arg and Di-Arg peptide species into insulin by carboxypeptidase B digestion and harvesting an insulin analog.
A process for preparing a human pro-insulin derivative comprising a C-peptide RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR, the process comprising:
- (a) preparing a pro-insulin peptide having a C-peptide amino acid sequence RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR;
- (b) modifying the peptide to include a met-histidine-Gly-Gly-Arg tag at the N-terminus of the peptide;
- (c) incorporating the nucleic acid sequence into an appropriate vector to provide a transformed vector;
- (pTrcHis vector);
- (d) transforming a population of competent cells comprising E. coli cells with the vector to provide transformed E. coli cells;
- (e) selecting transformed E. coli cells that express a peptide comprising an amino acid sequence -RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-AR;
- (f) culturing a composition of the selected transformed cells under conditions suitable for expression of the peptide having the amino acid sequence;
- (g) solubilizing the composition comprising the cells;
- and
- (h) purifying the human pro-insulin derivative from the culture.
Dependencies are inferred from the claim text.