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Insulin & TMOF_Yeast Comparison

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Insulin & TMOF_Yeast Comparison

  1. 1. Comparison of LMO Treatment in the Production of Insulin & TMOF_Yeast Use of LMO (E. Coli Bacteria) in Use of LMO (Pichia pastoris Yeast) to pharmaceutical Insulin production express TMOFTM in MOUSTIcideTM Production BamH I 5’ AOX1 T T PT EF Promoter Promoter 1 5 ’ A OX 1 β gal gene β gal gene pPICZ B PEM7 3.3 kb n Co E1 ci Insulin gene Insulin gene o Ze l A subunit B subunit c yc l T T Bgl IIAntibiotic Antibioticresistance gene resistance gene Transform into E. coli host TMOFTM Yeast A B TMOFTM molecule expressed within Pichia pastoris yeast cell β galactosidase / insulin A β galactosidase / insulin A fusion protein accumulates in cell fusion protein accumulates in cell 2 - Step LMO Elimination Process Extract and purify β gal/insulin fusion proteins LMO Treatment Heat Kill @ 75oC A B for 3 hours Treat with cyanogen bromide Purification 100% Drying with Heated Air Stream @ 235oC to cleave A and B chains Elimination for 3-5seconds A B Purify, mix A and B chains to form functional insulin 2 - Step LMO Kill Verification Disulfide bonds A 3 sets of petri dish culture simulation = culture 0 B Active Insulin 3 sets of shake ask culture simulation = culture 0 O PR LM ES ENT NO LMO 0% 10 KILLE D

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