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 II
Antibiotic Antibiotic
resistance 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