Preparation Of A Short C-peptide Insulin Glargine

Insulin as the specific medicine for diabetes mellitus treatment since eighty years ago.It is indispensable in treating the all types of diabetes,especially in diabete -related ketosis, acidosis, diabetic coma and maintain normal blood glucose. That's important in clinic.However, traditional short-acting insulin has no enough action time to require multiple injections each day, the patient compliance is poor; subcutaneous injection with absorption peaks; bedtime injection of increased risk of hypoglycemia at night shortcoming. Glargine as one of a long-acting insulins, with a daily injection of just once is able to maintain 24 hours; absorption is stabilize,no peak after absorption,reduce the occurrence of hypoglycemia and have good bioavailability, blood glucose control stability as advantages. Long-acting insulin is the future direction of development.Objectives: To construct Human Mini-proinsulin of PET32a-glargine containing the short C-peptide of EAEDKR by E. coli expression system. Screening high expression of origami (DE3) plysS and origami (DE3) re-engineered strain to obtain glargine recombinant proteins. The establishment production process of PET32a-glargine/origami (DE3) plysS and PET32a-glargine/origami (DE3) from the upstream to breaken stains, purification and digestion. To get the right structure, the purity of the higher activity of glargine.Methods:PET32a-glargine upstream build: 1) The whole gene synthesis contains a short C-peptide EAEDKR sequences. 2) the use of PET32a plasmid BamH I and Hind III restriction sites , inserted glargine into PET32a plasmid, build PET32a-glargine recombinant plasmid. 3) The recombinant plasmids were transformed into Jm109 engineered stain, screening high-copy plasmid. 4) The high-copy plasmids were transformed into origami (DE3) plysS and origami (DE3) re-engineering bacteria. 5) The use of different selection pressures, inducing agent, induction method to obtain high expression of origami (DE3) plysS and origami (DE3) strain. 6) The optimal conditions and fermentation by fermentation.â‘¡PET32a-glargine/origami (DE3) plysS and PET32a-glargine/origami (DE3) bacteria-breaking technology research: testing at different PH and some kinds of broken bacteria Bacilli break conditions of the situation, choose breaking strain complete and mostly soluble exists methods in terms of broken bacteria.â‘¢Trx-glargine for purification and digestion: fermentation broth by Ni column and the Q column purification, EK enzymes and trypsin digestion, HPLC monitoring of digest effects . And to RP-C-18 column recovery, identified by mass spectrometry for structural identification.Results:â‘ SDS-PAGE, HPLC and mass spectrometry analysis of the molecular weight of glargine in line with the theoretical molecular weight.â‘¡PET32a-glargine/origami (DE3) expression was high, up to 1g / L.â‘¢Recovery rate is low, only 40%. Conclusion:This study achieved a short C-peptide containing the human insulin analogue glargine system in E. coli highly expressed through a series of purification and digestion end up with the target protein biological activity glargine. But the study found that although PET32a system make the higher expression of the target protein available, but some of them exists in a non-soluble, plasmid containing the Trx refolding of some help, recovery rate is higher than previously reported .