Download Molecular Evolution for Biotechnology

Molecular Evolution for
Biotechnology
Assistant Professor Dr. Montarop Yamabhai
School of Biotechnology,
Suranaree University of Technology
111 University Avenue, Nakhon Ratchasima, 30000.
THAILAND
Background
B.Sc. (Pharmacy), Mahidol University,
Thailand, 1989
Ph.D. (Biology), University
of North Carolina at Chapel
Hill, USA, 1998
Post Doc at U. of Texas
Southwestern Medical Center,
Dallas, TX, USA, 2001
Humboldt Fellow 2002-2003
Kai Simons [Director Emeritus]
Previous Research in USA and
Germany
Molecular Cell Biology involving the study
of protein-protein interaction in cellular
signaling and trafficking
• Identification of a protein called “Intersectin”, which involve
in endocytic and exocytic pathways
• Targeting of protein to membrane microdomain called lipid
rafts or caveolae
Current Research
• Application of “molecular evolution”
for Biotechnology
• 2 High-throughputs techniques
– Directed Evolution
– Phage Display Technology
Directed Evolution
Modern Evolutionary Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Mutation
Selection
Modern Evolutionary Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Selection
Selection
Selection
Selection
Selection
Modern Evolutionary Theory
RA Fisher, S Wright, JBS Haldane
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Selection
Selection
Selection
Selection
Directed Evolution
3.2 months/yrs
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Mutation
Mutation
Mutation
Mutation
Recombination
Recombination
Recombination
Recombination
Recombination
Selection
Selection
Selection
Selection
Selection
Recombination
Recombination
Recombination
Recombination
Recombination
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Mutation
Selection
Selection
Selection
Selection
Selection
Selection
Directed Evolution
Project I
Establish DNA shuffling
technology
BIOTEC, NSTDA
Directed Evolution
Phage Display Technology
What can be displayed?
•
•
•
•
•
Proteins from 6-300 aa
Short peptides
Antibody fragments
cDNA library
enzymes
1
Pan library with
immobilized targets
2
Wash off unbound phage
5
Pan eluted phage
3-4
3
Elute bound phage
4
Amplify overnight
© 2004 Montarop Yamabhai
6
Isolate individual colony
Project II
Construction of Phage Display Libraries
supported by NRCT
............
Random peptide (X)12
Other Projects
Supported by IFS, AUNP, TRF, NRCT
• Using Directed Evolution (DE) Technique
to improve the property of industrial
enzymes e.g.
– Chitinase
– Amylase
– Mannanase
• Selection of Phage-displayed peptides
and scFv against
- cell surface of
cholangiocarcinoma
(bile duct cancer)
- starch
Enzyme = White Technology
Improvement of Chitinase
Enzyme
Km
(mM)
pH6
pFChi13
pFChi8785
128mt
Vmax
(μmole/min/mg)
kcat
(s-1)
kcat/Km
(s-1/mM)
0.050±0.002
0.029±0.001
0.033±0.004
2.050±0.065
1.550±0.071
3.100±0.141
2.200±0.075
1.667±0.075
3.335±0.140
45.380±0.424
54.117±1.254
102.467±1.825
~2x
pH3
pChi13
0.098±0.003
pFChi8785 0.095±0.007
128mt
0.058±0.007
1.025±0.023
0.640±0.028
1.100±0
1.099±0.027
0.688±0.029
1.183±0
11.274±0.133
7.326±0.097
20.556±0.038
~2x
Future Project
• Try to understand the mechanism of
actions of selected Thai traditional
medicines
MY LAB
20 Nov 2007