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7.344 Directed Evolution: Engineering Biocatalysts
Spring 2008
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Alternative methods for library
generation
Lai, Y.-P.; Huang, J.; Wang, L.-F.; Li, J.; Wu, Z.-J. A new approach
to random mutagenesis in vitro. Biotech. Bioeng. 2004, 86, 622-627.
Coia, G.; Ayres, A.; Lilley, G.G.; Hudson, P.J.; Irving, R.A. Use of
mutator cells as a means for increasing production levels of a
recombinant antibody directed against Hepatitis B. Gene 1997, 201,
203-209.
Chemical mutagenesis
• How does
mutagenesis occur?
What do the authors
use this method to
study?
• What are the results?
• What are the benefits
to using this method?
• Are there any pitfalls
to this method?
O
O
H3C
S
O
EMS
Figure 1
Image remove due to copyright restrictions
Please see Fig. 1 in Lai, Y.-P, J. Huang, L.-F.
Wang, J. Li, and Z.-J. Wu. “A new approach to
random mutagenesis in vitro.” Biotech. Bioeng.
86(2004): 622-627.
EMS induced mutation
H2
N
O
N
N
NH
N
N
N
O
H2N
C
G
O
N
O
NH
N
NH
N
N
O
NHEt
G + EMS
T
Figure 2
Image remove due to copyright restrictions
Please see Fig. 2 in Lai, Y.-P, J. Huang, L.-F.
Wang, J. Li, and Z.-J. Wu. “A new approach to
random mutagenesis in vitro.” Biotech. Bioeng.
86(2004): 622-627.
Use of E. coli mutator strains
• What is the technique and how does it work?
• What are the authors trying to use this technique
for? What are the results? (Figs 4-6)
• How could you calculate the mutational
frequency for this method? (Hint: use Figure 4)
• What are the benefits to this method?
• What are the pitfalls?
Scheme of technique
pIII
TGA SECIS gIII
Kan
scFv
scFv
gIII
Helper Phage
Phage produced by
superinfection
Tet
Phagemid
ELISA against HepB
Surface antigen
Express finally selected clones in an
expressing strain – has a stop codon
between scFv and pIII
Superinfect for
panning
scFv
Phage selected by
ELISA
Mutator strain results
Image removed due to copyright restrictions.
Please see Figs. 4 and 5 in Coia, G, A. Ayres, G. G.
Lilley, P. J. Hudson, and R. A. Irving. “Use of mutator
cells as a means for increasing production levels of a
recombinant antibody directed against Hepatitis B.”
Gene 201(1997): 203-209.
For next week…genetic
complementation
Plasmid
Nutrient
Enzyme
Compound
Aspartate 2-oxoglutarate
aminotransferase
Cephalosporin acylase
H
N
H 2N
S
O
CO2H
CH3
N
CH3
O
CO2H
Adipate feed
Acyltransferase
H
N
S
O
CO2H
N
CH3
CH3
O
CO2H
Expandase/
Hydroxylase
Expandase
H
N
O
CO2H
H
N
S
N
O
CO2H
O
S
N
O
CH3
CH3OH
CO2H
CO2H
Acetyltransferase
Acylase
H
N
S
O
CO2H
O
N
O
CH2OCCH3
CO2H
H2N
S
Acylase
O
N
O
CH2OCCH3
H 2N
S
CO2H
O
N
O
CH2OCCH3
CO2H