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: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