Enhancement of recombinant monomeric insulin production in methylotrophic yeasts by increasing copy number of gene : Completed research report (2nd year)

Abstract

Insulin is a hormone that is produced in pancreas. It is important for regulation of blood sugar level. The active form of insulin is monomer which contains 51 amino acids in two peptide chains. Currently, improvement of insulin production was done by using recombinant DNA technology in bacteria and yeasts. The obtained recombinant insulin is easier to be purified than the pancreatic insulin. This research has been using the recombinant yeast, Pichia pastoris KM71H (TP1), which has a cassette of monomeric insulin precursor (MIP) to produce recombinant MIP. The recombinant MIP was induced to be expressed by 0.5% methanol in MMH medium. The recombinant MIP expression was detected and quantitatively determination by dot-blot analysis and indirect competitive ELISA, respectively. The expression level of recombinant MIP was 16 mg/L. The molecular weight of MIP that was determined by MALDI-TOF mass spectra technique is 5756.95 Da. The recombinant MIP was purified from culture broth by two steps, i.e. SP sepharose fast flow cation exchange chromatography and 10 kDa Amicon centrifugal molecular weight cutoff. Afterwards, it was converted to active form by TPCK tryptic hydrolysis. The tryptic digested MIP was used to test the activity through the expression of glucose transporter 4 (GLUT4) gene in H9c2 (2-1) rat myocardial cell line by real-time PCR. The results showed that the active MIP (11.20 ug/L) induced the expression of GLUT4 gene and the glucose in culture medium of H9c2 (2-1) cell line significantly reduced when the cell line was treated with the active MIP at the concentration 0.70 to 11.20 ug/L.