The document compares using LMO (E. coli bacteria) and LMO (Pichia pastoris yeast) to produce insulin and TMOFTM. E. coli is used to produce insulin by transforming it with genes for insulin subunits A and B, while Pichia pastoris is used to express the TMOFTM molecule. Both production methods involve a two-step LMO elimination process using heat and drying to kill the LMOs, verified by culture simulations showing no LMO presence.

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