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 & Chemiluminescence Lyon, France April 21, 2010

rapid inactivation of the wild-type luciferase at elevated temperatures often limits its applications [E]=0,01 mg/ml 50 mM Tris-acetate, 20 mM MgSO 4, 2mM EDTA, 0,2 mg/ml BSA, pH 7.8 1/2 =67 min 1/2 =9 min Irreversible thermal inactivation of the wild-type L. mingrelica luciferase

Random PCR mutagenesis error-prone PCR residues of luciferase --- 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 ( residues) error-prone PCR 4 cycles of mutagenesis thermostable mutants library of E. coli colonies

After 40 min at 50°CBioluminescence after growth of the cells

Four cycles of mutagenesis led to the mutant 4TS S118C1TM1 37˚C 50˚C 2TM1 50˚C 3TM1 55˚C 4TS T213S S364C K156R A217V C146S E356K R211L 1TM1 WT 3TM1 3TM2 3TM3 2TM1 in vivo bioluminescence of E. coli colonies after 40 min at 50°C

Kinetic properties

Bioluminescence spectra I/I max Effect of pH (at 25°C) Effect of temperature

Irreversible thermal inactivation of 4TS and WT luciferase Conditions: 50 mM Tris-acetate, 20 mM MgSO 4, 2 mM EDTA, 0,2 mg/ml BSA, pH 7.8, 0,01 mg/ml luciferase

Comparison of thermal inactivation in different conditions 50 mM Tris-acetate, 20 mM MgSO 4, 2 mM EDTA, 0,2 mg/ml BSA, pH mM Na-phosphate, 410 мМ (NH 4 ) 2 SO 4, 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) 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.

Research support RFBR grant

Supporting slides: An electronic version of this presentation with commentaries can be downloaded here:

Mutants obtained during mutagenesis

After 20 min After 40 min 3TM1 4TS In vivo bioluminescence after incubation of colonies at 55°C: mutants 3TM1 and 4TS

Expression plasmid pETL7 luc-AKM Native luciferase ApaI restriction site MASK~luc-SGPVEHHHHHH

WT S118C 1TM1 2TM1 screening 3TMx