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:koksharov83@ya.ru

Research supportRFBR grant 08-04-00624

e-mail:koksharov83@ya.ru

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