Download - Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution
Thermostability enhancement of
Luciola mingrelica firefly luciferase
by in vivo directed evolution
Koksharov M.I., Ugarova N.N.
Department of Chemistry,Lomonosov Moscow State University
16th Symposium on Bioluminescence & ChemiluminescenceLyon, France April 21, 2010
rapid inactivation of the wild-type luciferase at elevated temperatures often limits its applications
[E]=0,01 mg/ml50 mM Tris-acetate, 20 mM MgSO4,2mM EDTA, 0,2 mg/ml BSA, pH 7.8
0 50 100 150 2000
25
50
75
100
37°C
Activity, %
time, min
42°C
1/2=67 min
1/2=9 min
Irreversible thermal inactivation of the wild-type L. mingrelica luciferase
Random PCR mutagenesis
pLR36270 bp
LuxR
LuxI
люцифераза
bla
ROP
pr
ori
мутитруемый участок
Bam HI (2290)
Bgl II (2787)
Nhe I (1607)
Xho I (2002)
Afl II (3236)
error-pronePCR
130-390 residues of luciferase
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luciferase
1) incubation of colonies at 37-55°C
2) selection of clones with the highest
in vivo bioluminescence
The scheme of mutagenesis and screening
luciferase gene library of mutant genes
subcloning,transformation
Random mutagenesis(130-390 residues)
error-prone PCR
4 cycles ofmutagenesis
thermostablemutants
library of E. colicolonies
After 40 min at 50°CBioluminescence after growthof the cells
Four cycles of mutagenesis led to the mutant “4TS”
S118C 1TM137˚C 50˚C
2TM150˚C
3TM155˚C
4TS
T213SS364C
K156RA217V
C146SE356K
R211L
1TM1 WT
3TM13TM23TM3
2TM1
in vivo bioluminescence of E. coli coloniesafter 40 min at 50°C
Kinetic properties
max (half-width), nm Km, μМ
Specific activity, % WT
LH2 ATP pH 7.8 pH 6.0
1/2, min (42°C)
WT 100 72±5 330±40 566 (78) 616 (79) 9,1
4TS 190 60±5 41±8 573 (92) 609 (91) 592
Bioluminescence spectra
500 550 600 6500,0
0,5
1,0
, nm
4TS
pH 7.8
4TSWT
pH 6.0
WT
500 550 600 6500,0
0,5
1,0
, nm
25°C
10°C
4TS
4TS
WT
WT
I/Imax
Effect of pH (at 25°C)
Effect of temperature
Irreversible thermal inactivation of 4TS and WT luciferase
0 10 20 30 40 50-4
-3
-2
-1
0
WT, 37°C
WT, 42°C
4TS, 42°C
4TS, 37°C
ln(A/A0)
time, hours
Conditions: 50 mM Tris-acetate, 20 mM MgSO4, 2 mM EDTA, 0,2 mg/ml BSA, pH 7.8, 0,01 mg/ml luciferase
Comparison of thermal inactivation in different conditions
half-life, hours
37 °C 42 °C 45 °C 50 °C
WT 1,1 0,15 0,047 0,01
4TS 128 10 0,72 0,062
1/2 increase
116 67 15 6
half-life, hours
37 °C 42 °C 45 °C 50 °C
WT 33 0,95 0,21 0,037
4TS n.d. n.d. 33 0,74
increase
- - 157 20
50 mM Tris-acetate, 20 mM MgSO4, 2 mM EDTA, 0,2 mg/ml BSA, pH 7.8
50 mM Na-phosphate, 410 мМ (NH4)2SO4, 2 mM EDTA, 0,2 mg/ml BSA, pH 7.8
Localization of the substitutions of 4TS in the luciferase structure
Summary
1) E. coli cells remain viable after incubation at temperatures up to 55°C and after in vivo bioluminescence detection. Thus thermostable mutants of luciferase can be produced in a simple and rapid manner by a non-lethal in vivo screening of mutant colonies for thermostability.
2) 130-390 residues of Luciola mingrelica firefly luciferase were subjected to 4 cycles of directed evolution which led to the mutant designated “4TS” with a significantly improved thermostability.
3) The half-life at 42°C increased 66-fold from 9 minutes to about 10 hours. The specific activity increased almost 2-fold.
4) 4TS retains 70% activity after two days of incubation at 37°C so its stability is sufficient for most common applications.
e-mail:[email protected]
Research supportRFBR grant 08-04-00624
e-mail:[email protected]
Research supportRFBR grant 08-04-00624
Supporting slides:
An electronic version of this presentation with commentariescan be downloaded here:
http://koksharov83.narod.ru/docs/lab/ISBC-2010_Koksharov_presentation.ppt
http://koksharov83.narod.ru/docs/lab/presentations.html