INSULIN PRODUCTION METHODS AND PRO-INSULIN CONSTRUCTS
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.
1 .- 8 . (canceled)
9 . 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.
10 . The process of claim 9 , wherein the E. coli is a BL21 production E. coli.
11 . The process of claim 9 , wherein the peptide is further defined as comprising an amino acid sequence encoding a substituted human pro-insulin derivative protein having an amino acid sequence comprising:
MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVG
QVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCN.
12 . The process of claim 9 , wherein the reverse phase chromatography column is further defined as comprising a silica based media with a C4, C8, or C18 bonded phase.
13 . The process of claim 9 wherein the suitable vector is a pTrcHis2A (Kan) vector.
14 . A pharmaceutical product comprising recombinant human insulin prepared by the process of claim 9 .
15 . 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.
16 . The process of claim 15 , further comprising the step:
(i) crystallizing the human pro-insulin derivative.
17 . The process of claim 15 , further comprising preparing human insulin from the human pro-insulin derivative by enzymatic hydrolysis.
18 . The process of claim 15 wherein the E. coli is a BL21 production E. coli.
19 . A composition prepared by the process of claim 17 , wherein the composition is essentially free of human pro-insulin.