Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution
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Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution

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Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution...

Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution

16th International Symposium on Bioluminescence and Chemiluminescence; Lyon, France; April 21, 2010

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  • 16th Symposium on Bioluminescence & Chemiluminescence Lyon, France April 21, 2010 --------------------------------- Thermostability enhancement of Luciola mingrelica firefly luciferase by the approach of in vivo directed evolution
  • Due to the high quantum yield of bioluminescence, catalytic activity and substrate specificity this enzyme is widely used in methods based on ATP determination, as a luminescent biomarker in molecular biology and in a number of other applications. However, rapid inactivation of the wild-type luciferases at temperatures above 30°C often limits their current and potential applications. Luciferase from Luciola mingrelica fireflies displays low thermostability loosing about 50% of activity within an hour at 37°C and within 9 minutes at 42°C. According to the literature various approaches have been used to increase the thermostability of firefly luciferases. The relative stability at 37°C can be achieved by the addition of stabilizing compounds ( Eriksson et al., 2003; Mehrabi et al., 2008; Moroz et al., 2008) . But the mutagenesis approach appears to be the most successful ( Kajiyama et al., 1993; White et al., 1996; Hall et al., 1998; Kitayama et al., 2003) , especially random one, since bioluminescence allows conducting of a simple screening for residual activity.
  • In this work consecutive cycles of random PCR mutagenesis were used to significantly increase luciferase thermostability. The mutant S118C was used as a starting point due to its slightly higher thermostability than that of the wild type enzyme. Mutagenesis was applied to a part of the gene coding 130-391 out of 548 amino acid residues of L. mingrelica luciferase. This region is shown in magenta in both the gene and the protein 3D-structure. See the slide.
  • Four consecutive rounds of random PCR mutagenesis and screening were carried out according to the following scheme. First the library of mutant genes is obtained by error prone PCR of the part of the gene of luciferase. The required gene region is subcloned into an expression vector and transformed to E. coli cells. This gives us a library of colonies producing different mutants of luciferase. Then they are screened for an increased thermostability by two steps. The most stable mutant obtained can be used in the following cycle of mutagenesis. At the first step of screening plates with the colonies are incubated at elevated temperatures up to 55°C. This leads to the inactivation of unstable forms of luciferase produced in the cells.
  • Then the plates are treated with a luciferin solution to identify thermostable mutants since they retain a high level of in vivo bioluminescence. See the slide . Because the cells are not killed during this type of screening it eliminates the need of replica plates and significantly simplifies the procedure allowing us to carry out each cycle of mutagenesis in a rapid and efficient manner.
  • Four consecutive rounds of random PCR mutagenesis and screening were conducted. It led to a significant improvement of luciferase thermostability without compromising its activity. The resulting mutant was designated 4TS. This photograph illustrates the gradual increase of the in vivo thermostability of the mutants with each cycle of mutagenesis. The mutant 4TS contains 8 point mutations relative to the wild-type enzyme. The order of appearance of these mutations is shown in this scheme. The analysis of substitutions shows that 4 mutations (in bold) are mainly responsible for the increased thermostability: R211L, A217V, E356L and S364C. Thermostabilizing mutations of A217 and E356 residues are well known for a number of firefly luciferases (Kajiyama, Nakano, 1993; White et al., 1996). Mutations of R211 and S364 residues are reported for the first time.
  • Kinetic and bioluminescent properties of the mutant 4TS were studied as well as its thermal inactivation at different conditions. The main results are presented in this table. The mutant has a half-life at 42°C of about 10 h, a 66-fold increase over the wild-type enzyme. Moreover, the specific activity increased almost 2-fold. The Michaelis constant for ATP became 8 times lower which can be useful in ATP determination.
  • Bioluminescence spectra of the mutant are wider than that of the wild-type. This widening almost disappears at low temperatures so it corresponds to the faster increase of the red spectral component with temperature and not to the higher pH-sensitivity.
  • As can be seen from this figure the thermostability of the mutant 4TS increased dramatically: from 9 minutes to about 10 hours at 42°C. While the half-life of the wild-type luciferase is about an hour at 37°C the mutant in this conditions retains 70% activity after two days of incubation. Since firefly luciferase is usually used at temperatures up to 37°C, this level of stability is sufficient for most common applications.
  • The kinetics of thermal inactivation of the wild-type and mutant luciferase was compared at different temperatures in two buffer solutions. We commonly study thermostability in the tris-acetate buffer (Koksharov, Ugarova, 2008) which is close to the composition of our ATP reagents (Moroz et al., 2008). For comparison, thermal inactivation was studied in a sodium phosphate buffer solution of the following composition since it was used in a number of works on luciferase mutagenesis ( Kajiyama et al., 1994; White et al., 1996; Kitayama et al., 2003) . As can be seen this buffer solution causes significant stabilization at all temperatures studied. So the effect of different mutations can be compared only in similar buffer conditions. Comment: 0,2 mg/ml BSA is added in order to prevent the influence of adsorption during a long-term incubation of the thermostable mutants.
  • This slide shows the location of substitutions of the mutant 4TS in the structure of luciferase. 4 key substitutions are shown in yellow. In most of these cases the increase in thermostability is caused by the substitution of internal polar residues with hydrophobic ones (S118C, R211L, S364C) or by filling of the internal cavity with a larger hydrophobic group (A217L). It can be noted that the key mutations are located in the second subdomain of luciferase. The substitution S118C is located in the interface of this subdomain with another. According to the literature the second subdomain is much less stable than the other two (Frydman et al., 2000). So it’s not surprising that the mutations obtained in this study as well as most of the thermostabilizing mutations described in the literature tend to be located in this subdomain or its interfaces.
  • Thank you for your attention! A photo of Moscow State University Main Building . In front there is a monument of M.V. Lomonosov .
  • Thank you for your attention! A photo of Moscow State University Main Building . A winter view. :)
  • 4TS shows the highest in vivo thermostability among the other mutants obtaied in this work.
  • The expression vecor and the corresponding form of luciferase.
  • A screening of one of the plates during the 3th cycle of mutagenesis.

Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution Thermostability enhancement of Luciola mingrelica firefly luciferase by in vivo directed evolution Presentation Transcript

  • 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 130-390 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 (130-390 residues ) error-prone PCR 4 cycles of mutagenesis thermostable mutants library of E. coli colonies
  • After 40 min at 50 °C Bioluminescence after growth of the cells
  • Four cycles of mutagenesis led to the mutant “4TS” S118C 1TM1 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 7.8 50 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
    • 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.
    • 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.
    • The half-life at 42°C increased 66-fold from 9 minutes to about 10 hours. The specific activity increased almost 2-fold.
    • 4TS retains 70% activity after two days of incubation at 37°C so its stability is sufficient for most common applications.
  • e-mail: [email_address] Research support RFBR grant 08-04-00624
  • e-mail: [email_address] Research support RFBR grant 08-04-00624
  • Supporting slides: An electronic version of this presentation with commentaries can be downloaded here: http://koksharov83.narod.ru/docs/lab/ISBC-2010_Koksharov_presentation.ppt http://koksharov83.narod.ru/docs/lab/presentations.html
  • Mutants obtained during mutagenesis
  • After 2 0 min After 4 0 min 3TM1 4TS 4TS In vivo bioluminescence after incubation of colonies at 5 5 °C: mutants 3TM1 and 4TS
  • Expression plasmid pETL7 luc -AKM Native luciferase ApaI restriction site MASK ~luc - S GP VE HHHHHH
  • WT  S118C  1TM1  2TM1  screening 3TMx