Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence

Thermostability of Firef ly Luciferases Affects Efficiency ofDetection by In Vivo Bioluminescence

Brenda Baggett1 Rupali Roy1 Shafinaz Momen1 Sherif Morgan1 Laurence Tisi2

David Morse1 and Robert J Gillies1

1University of Arizona and 2Cambridge University

AbstractLuciferase from the North American firefly (Photinis pyralis) is

a useful reporter gene in vivo allowing noninvasive imaging of

tumor growth metastasis gene transfer drug treatment and

gene expression Luciferase is heat labile with an in vitro half-

life of approximately 3 min at 37C We have characterized wild

type and six thermostabilized mutant luciferases In vitro

mutants showed half-lives between 2- and 25-fold higher than

wild type Luciferase transfected mammalian cells were used to

determine in vivo half-lives following cycloheximide inhibition

of de novo protein synthesis This showed increased in vivo

thermostability in both wild-type and mutant luciferases This

may be due to a variety of factors including chaperone activity

as steady-state luciferase levels were reduced by geldanamycin

an Hsp90 inhibitor Mice inoculated with tumor cells stably

transfected with mutant or wild-type luciferases were imaged

Increased light production and sensitivity were observed in the

tumors bearing thermostable luciferase Thermostable pro-

teins increase imaging sensitivity Presumably as more active

protein accumulates detection is possible from a smaller

number of mutant transfected cells compared to wild-type

transfected cells Mol Imaging (2004) 3 324ndash 332

Keywords Luciferase in vivo bioluminescence thermostability breast cancer tumors

metastasis

Introduction

In the current study we test the hypothesis that ther-

mostabilization will lead to a higher accumulation of

firefly luciferase in human tumor cells and that this will

improve the lower detection limit of luciferase express-

ing cells in whole animals Luciferase is a powerful

in vivo reporter system whose main strength is the rela-

tive absence of endogenous light-emitting signals from

higher organisms Hence it can provide extremely high

signal to noise Over the past few years methods have

been developed to detect expression of luciferase pro-

teins in living mice [1ndash3] Applications of this technol-

ogy have grown and now include the detection of

tumor growth [4] the detection of tumor metastases

[5] as a reporter for gene expression under control of

regulable promoters [6ndash9] as a reporter for efficacy of

gene therapy [810ndash13] measurement of in vivo phar-

macodynamics [1415] and as a reporter for toxicology

studies [16ndash18]

Luciferases are made by a wide variety of phyla in-

cluding bioluminescent species such as beetles fireflies

bacteria and marine coelenterates and dinoflagellates

[19ndash24] The most widely used luciferase reporter gene

is that of the North American firefly Photinis pyralis

Wild-type P pyralis luciferase is thermolabile with an

in vitro half-life for activity on the order of 2ndash3 min at

37C (see Results) However luciferase is stabilized

in vivo with half-lives from 1 to 4 hr [2526] This has

certain obvious advantages as well as disadvantages On

the one hand a high turnover rate of the reporter would

be advantageous in time-sensitive studies such as those

to determine the response to an environmental toxin

infection or induction or repression of gene expression

However a high turnover rate could limit the accumula-

tion of functional luciferase molecules and would un-

doubtedly have a higher explicit energy cost associated

with continuous de novo protein synthesis On the other

hand it is proposed that a lower turnover rate provided

by thermostabilization will lead to higher accumulation of

active luciferase molecules and that this will lead to

higher light output on a per-cell basis and thus improve

detection This would be advantageous for studies that

are not time-sensitive such as monitoring tumor growth

or colonization of metastases This is especially important

in investigating metastases as there would be significant

interest in the fate of micro-metastases which might

otherwise escape detection due to their small size

Materials and Methods

Chemicals and Buffers

Unless otherwise noted all chemicals were obtained

from Sigma (St Louis MO) Hanks Balanced Salt Solution

D 2005 Massachusetts Institute of Technology

Corresponding author Robert J Gillies University of Arizona Cancer Center Tucson AZ

85724-5024 e-mail gilliesemailarizonaedu

These authors contributed equally to this work

Received 10 November 2003 Accepted 17 August 2004

RESEARCH ARTICLE Molecular Imaging Vol 3 No 4 October 2004 pp 324 ndash 332 324

(HBSS) 54 mM KCl 04 mM KH2PO4 42 mM NaHCO3

137 mM NaCl 03 mM Na2HPO4 In order to gener-

ate HBSS at different pH values pH was adjusted by

adding either HCl or NaOH to the desired pH Native

firefly luciferase-recombinant protein was purchased

from RampD Systems (Minneapolis MN) (700-LF-01M)

LuciferinmdashD-luciferin potassium salt was a generous gift

from Brian Ross (University of Ann Arbor Michigan)

Luciferase Assay reagent (LAR) and Cell Culture Lysis Re-

agent (CCLR) were purchased from Promega (Madison

WI) Luciferase mutants were cloned into a pET23a(+)

bacterial expression vector at the NdeI and SalI sites of

the MCS and maintained in Escherichia coli B121 DE3

Wild-type luciferase was isolated from pGEM-luc from

Promega The mammalian expression vector used was

pcDNA31() from Invitrogen (Carlsbad CA) with a se-

lectable neomycin resistance gene

Production of Mutant Luciferases from Bacteria

Preliminary experiments were done to optimize for

induction time DH5a bacteria were transformed with a

pET23a(+)mutant luciferase construct Colonies of

bacteria were isolated then grown in liquid cultures of

LB broth (Sigma L 3152) in an orbital shaking incubator

One milliliter of cell suspension was removed the bacte-

rial cells pelleted then resuspended in 1 mL HBSS

pH 78 This sample was placed in the luminometer a

background reading was taken D-luciferin was added

and the light intensity was recorded IPTG was then

added to the remainder of the culture to a final concen-

tration of 01 mM and cultures were left in an orbital

shaking incubator Samples were then removed at multi-

ple time points post-IPTG induction and the assay pro-

cess was repeated Peak induction was seen between

1 and 2 hr In vitro experiments were performed by

isolating the mutant luciferase protein from bacterial

cultures In an orbital shaking incubator 2-mL starter

cultures were grown from isolated colonies incubated

overnight and then transferred to 100 mL LB broth After

incubating for 2 hr cells were induced with IPTG Two

hours post-induction cells were harvested and washed a

lysozyme buffer (9 mgmL lysozyme in PBS) was added

and the cells were subjected to a freezendashthaw cycle after

addition of Triton X-100 Samples were spun and super-

natant aliquoted then frozen for use with in vitro tem-

perature half-life assays

Mammalian Cell Culture

All mammalian cell lines were grown in DMEF-12

media (Sigma D0547) with 10 FBS Cells were pas-

saged weekly The parental cell lines MDA-mb-231

(metastatic breast) and SW-480 (colon) were plated into

six-well plates or 10-cm dishes for transfections Stable

cell lines were created by adding selection media (G418

sulfate Geneticin 400 mgmL Invitrogen) and cells

maintained were under selection Cells for mouse inoc-

ulations were expanded equally into several T-75 flasks

one flask was trypsinized and counted in an appropriate

number of flasks cells were scraped centrifuged and

resuspended in a volume of 11 salinecell suspension to

Matrigel (BD Biosciences Franklin Lakes NJ) so that a

100-mL inoculum contains 3 106 cells

Preparation of Mammalian Expression Vectors

DNA was isolated from colonies of B121 DE3 E coli

transformed with a bacterial expression vector carrying

one of six different mutant forms of luciferase that were

previously generated by a random mutagenesis screen in

yeast These mutant sequences were cloned into the

NdeI and SalI sites of the MCS of pET23a(+) (Tisi

unpublished) Competent DH5a cells were then trans-

formed with the various mutants designated A-F (see

Table 1) Each mutant DNA was isolated digested and

ligated into a mammalian expression vector All mutants

were transferred into the pcDNA31() mammalian

expression vector at ApaI and XhoI The wild-type

luciferase was transferred from pGEM-luc (Promega) to

pcDNA31() also at ApaI and XhoI SW-480 colon can-

cer cells were transiently transfected using FuGene-6

Table 1 Thermostability of Luciferases

Luciferase Mutations In vitro In vivo

T05 at 37C DG+ (kJmol) T05 at 37C

WT NA 306 36190 493 plusmn 214

Mutant A E345K T214A 85 plusmn 10 30973 1363 plusmn 297

Mutant B E345K I232A

T214A

155 plusmn 145 35340 1419 plusmn 157

Mutant C E345K A215L 736 plusmn 182 40365 1604 plusmn 353

Mutant D E345K I232A

T214A F295L

S420T

724 plusmn 31 27074 1281 plusmn 114

Mutant E E345K A215L

I232A T214A

F295L

821 plusmn 85 28068 2494 plusmn 587

Mutant F E345K A215L

I232A T214A

751 plusmn 981 36246 1355 plusmn 169

For in vitro studies luciferase mutants were cloned expressed and purified

from DH5a bacteria and incubated at various temperatures (from 21C to 45C)

for various times prior to assay at 23C Plots of enzyme activity (light output)

versus time were fitted to first-order exponential to obtain half-lives Arrhenius

plot of half-lives versus temperature was generated for each mutant to

determine the DG+ associated with the inactivation process For in vivo

studies SW-480 cells stably expressing each of these luciferases were treated

with 001 mM cycloheximide at 37C and extracted at time points thereafter

The resulting plot of light output versus time was fitted to a first-order

exponential to yield half-life

Thermostable Luciferases Baggett et al 325

Molecular Imaging Vol 3 No 4 October 2004

(Roche Indianapolis IN) with each of the mammalian

expression constructs Transfected cells were incubated

for 48 hr lyzed with CCLR (Promega) and assayed for

light Polyclonal stable transfectants were also created

by transfection of SW-480 cells or MDA-mb-231 cells

using FuGene-6 48-hr incubation followed by selec-

tion using media containing 04 mgmL G418 Cells con-

tinued to be passaged and grown in selection media

All stable lines were tested to confirm light production

Quantitative RT-PCR was conducted on RNA extracts of

the various transfected cell lines to determine levels of

message being produced by each cell line

Detection of Light Output in Cells and Extracts

For most studies light output was detected in vitro

using an SLM 8100C spectrofluorometer operating with-

out excitation Photons were collected by Hamamatsu

photomultipliers operating at room temperature with

attendant dark current For all studies the PMT voltage

and gains were kept constant at 1250 V and 10

respectively allowing comparison between assays run

at different times The reproducibility was verified in

parallel experiments (data not shown)

Detection of Light Output In Vivo

Wild-type luciferase and thermostable mutants are to

be compared to each other by imaging the cells in mice

Mutant luciferase cells were chosen for imaging studies

based on mRNA expression levels being comparable to

wild-type mRNA expression as well as having a long

half-life at 37C Mutant F was chosen as the mutant

luciferase for these studies SCID mice were inoculated

subcutaneously with MDA-mb-231WT on the left flank

and MDA-mb-231mut F on the right flank using 3 106 cells in 100 mL Matrigel On Day 8 tumors were

barely palpable The mice were anesthetized using a Ke-

tamine Acepromazine and Xylazine cocktail (72 mgkg

Ketamine 6 mgkg each Acepromazine and Xylazine)

then imaged using a VersArray 1300B cooled CCD cam-

era (Roper Scientific Tucson AZ) A top-illuminated im-

age was taken using a 100-msec exposure at f 16 The

luminescent image was then taken using a 10-min expo-

sure at f 28 5 min after an intraperitoneal injection of

175 mgkg luciferin An intensity map was created using

WinView32 software (Princeton Instruments Trenton

NJ) The final image was created by superimposing the

color intensity map onto the light image using Adobe

Photoshop 60 The imaging process was repeated on

Days 15 22 and 29 and caliper measurements taken of

all tumors each time (tumors were only palpable not

measurable until Day 22)

qRT-PCR

Quantitative RT-PCR was done by isolating RNA from

cell lines using a tRNA isolation mini-prep kit (Sigma

RTN-70) DNase treatment (Ambion Austin TX Cat

1906) of the samples and followed by a SuperScript one-

step RT-PCR reaction (Invitrogen) The RT-PCR reaction

was run using a Smart Cycler (Cepheid Sunnyvale CA)

Reactions were set up using previously designed PCR

primers for luciferase (forward-GGGATACGACAAGGA-

TATGGGC reverse-TGGAACAACTTTACCGACCGC) and

a GAPDH control and detected by SYBR Green dye

(Molecular Probes Eugene OR) Further experiments

were carried out using a primerprobe design Primers

and a TET-labeled probe for luciferase (forward-GGC-

GCGTTATTTATCGGAGTT reverse-TGGCGAGGGTGCT-

TACGT probe-TET-TTGCGCCCGCGAACGACATT) were

designed by using Primer Express software (Applied

Biosystems Foster City CA) A commercially available

FAM-labeled b-actin primer and probe set (Applied Bio-

systems) were used as an internal control Data were

expressed relative to glyceraldehydes phosphate dehy-

drogenase (GAPDH) or b-actin mRNA Both of these have

been shown to be robust control mRNA for these cell

lines (Morse et al submitted)

Results

Determination of Optimal pH and Temperature of

Wild-Type Luciferase

The activity of native (wild-type) firefly luciferase is

sensitive to both pH and temperature Optimum pH was

determined by incubating 20 mL of recombinant lucifer-

ase in 880 mL of HBSS adjusted to various pH levels

Baseline PMT current was monitored until stable at

which time 100 mL of LAR was added and the change in

light output was recorded in arbitrary units Light output

was highest with the HBSS between pH 7 and 9 When

these data were corrected for final pH the peak range

decreased significantly as shown in Figure 1A Conse-

quently HBSS at a pH of 78 prior to addition of LAR was

used for all subsequent studies as the pH of the solution

did not change upon addition of LAR

The effect of temperature on luciferase activity is il-

lustrated in Figure 1B which shows the time-dependent

light output following addition of luciferase and LAR to

HBSS and assayed at 23C and 40C As shown in this

figure light output remains constant at 23C whereas

less light is transiently emitted from the incubation at

40C The thermosensitive component was determined

by independently incubating LAR HBSS and luciferase

at various temperatures prior to analysis Neither LAR

nor HBSS showed any thermolability (data not shown)

326 Thermostable Luciferases Baggett et al


Figure 1 Characterization of wild-type firefly luciferase in vitro and in vivo (A) pH sensitivity Commercial firefly luciferase was incubated at 23C in HBSS at

various pH values and LAR induced light output was measured Data are expressed as arbitrary units of bioluminescence plusmn SD (n 3 per datum) (B) Time course of

light output at 23C and 40C Luciferase and LAR were added to HBSS (pH 78) prewarmed to the indicated temperature and light captured by PMTs Data are

expressed as arbitrary units of bioluminescence Gains PMT voltages and volumes were identical between samples At 23C light was at a high intensity and remained

stable At 40C intensity was not as high initially and degraded over time (C) Decrease in light output from as a function of time at different temperatures Luciferase

was incubated at the indicated temperatures for the indicated amount of time after which it was cooled to 23C and assayed for light output by the addition of LAR

Data are expressed as arbitrary units of bioluminescence plusmn SD (n 3 per datum) (D) Half-lives at different temperatures with Arrhenius plot The half-lives of

luciferase were determined at different temperatures by exponential fit to data shown in C An inverse plot (inset) indicates an energy of activation of approximately

340 kJmol (E) Light output from cells following incubation at 27C and 37C Equivalent cultures of 9L glioma cells were incubated for 18 hr at either 23C or 37C

Cells were trypsinized and suspended in a cuvette containing HBSS at 23C For detection of in vivo luminescence luciferin was added to the cell suspension and the

luciferin induced light emission determined by PMTs Alternatively the in vitro luciferase activity was determined following lysis of the cells and addition of LAR Data

are expressed as arbitrary units of bioluminescence plusmn SD (n = 4 per datum)



However as shown in Figure 1C luciferase itself was

significantly thermolabile Luciferase was incubated at

different temperatures for various amounts of time in

HBSS (pH 78) after which the temperature was re-

duced to 23C and the increase in light output was mea-

sured in response to addition of LAR As shown in this

figure light output decreased with incubation time at

all temperatures The rates of decrease were multi-

phasic suggesting complex mechanisms yet were uni-

formly faster at higher temperatures Luciferase activity

was still present at up to 6 hr at 28C and 3 hr at 30C At

34C and 37C however luciferase activity was com-

pletely abolished by 50 and 20 min respectively From

these data half-lives were calculated by fitting the data to

simple first-order exponentials The half-lives at different

temperatures are shown in Figure 1D An inverse plot of

these data is shown in the inset These data were fit to

the Arrhenius equation k = A exp(EART) where k is the

rate of enzyme inactivation E is the activation energy

associated with the inactivation process in kJ per mole

and T is the absolute temperature in Kelvin This yields a

DGDagger activation energy of 3619 kJmol

An initial test was then conducted to assess whether

the thermolability of luciferase significantly affected

steady-state enzyme activity in vivo Cultures of 9L glioma

cells stably expressing WT luciferase were split and incu-

bated in parallel for 18 hr at 23C or 37C with the

expectation that lower temperatures would result in an

accumulation of enzyme At the end of the incubation

luciferase activity was assessed both in vivo with the

addition of luciferin to whole cells and following cell lysis

with the addition of LAR As shown in Figure 1E luciferase

levels increased significantly upon incubation at 23Ccompared to 37C supporting the hypothesis that lucif-

erase thermolability significantly affects steady-state en-

zyme accumulation in vivo

Thermostable Mutants

The previous data suggest that the thermolability of

luciferase could have a significant effect on steady-state

protein levels in vivo To further investigate this phe-

nomenon a series of thermostable mutants were ana-

lyzed (listed in Table 1) These were expressed and

isolated from DH5a bacteria and the in vitro half-lives

were determined as described in Materials and Methods

As shown in Table 1 and Figure 2B the in vitro half-lives

of these mutants were significantly longer as compared

to wild-type luciferase Note that the activation energy

DGDagger of enzyme inactivation remained between 280 and

404 kJmol for all mutants comparable to the 362 kJmol

for wild type

In Vivo Turnover Rates

A more detailed analysis of the in vivo significance

of thermolability was performed by determination of

in vivo protein turnover rates In these experiments

mutant luciferases were subcloned into mammalian

expression vectors (pcDNA31()) and stably expressed

in SW-480 colon carcinoma cells under selection with

G418 These cells were treated at 37C with 10 mM cy-

cloheximide which is sufficient to inhibit de novo pro-

tein synthesis The amount of active luciferase was

determined in vitro at different time points and the

subsequent rate of light reduction was used to estimate

the in vivo turnover rate A typical response is shown in

Figure 2C which shows the loss of light following

cycloheximide treatment of SW-480 cells expressing

luciferase mutant F As shown in this figure the light

decreased with first-order exponential kinetics with a

calculated half-life of 135 min Independent half-life

determinations (with n = 1 sample per time point)

were repeated at least four times per mutant and these

data were averaged A summary of results from all

mutants is presented in Table 1 Note that the in vivo

half-lives are significantly longer than those observed

in vitro and that in general the relative in vivo half-lives

compare favorably to the relative in vitro half-life values

(Figure 2B) In other words wild-type luciferase has the

shortest half-life and mutant E has the longest half-life

both in vitro and in vivo Western blot analyses show

higher luciferase protein levels therefore lower detec-

tion limits in mutant luciferase compared to wild-type

cell extracts (data not shown) Although the mech-

anisms behind the differences between in vivo and

in vitro half-lives are not known a reasonable hypothesis

is that this is due to the presence of chaperone proteins

in vivo [2728] This is supported by the observation that

geldanamycin an inhibitor of the chaperone Hsp90

leads to a reduction in steady-state wild-type luciferase

levels (Figure 2D) It should be noted that at this dose

(200 nM) geldanamycin is not toxic to MDA-mb-231

cells (data not shown) Therefore the decreased lucif-

erase activity was not due simply to cell death

In Vivo Imaging

To test the hypothesis that thermostabilization of

luciferases will lead to higher steady-state light output

mice were inoculated with tumor cells expressing either

wild-type or mutant luciferases In order for this com-

parison to be valid the steady-state luciferase mRNA

levels must be similar between two cell lines that are to

be compared SW-480 and MDA-mb-231 cells were trans-

fected with mutant and wild-type luciferases and mRNA



levels were compared by quantitative RT-PCR In initial

experiments primers for GAPDH were used to compare

luciferase levels by taking the log2 amount of luciferase

mRNA expressed relative to the log2 GAPDH product

from the same preparation Subsequent experiments

used a primerprobe design to assess by multiplex

analysis b-actin and luciferase levels within a single

reaction Steady-state luciferase mRNA levels were com-

pared across all cell lines and there was a general

agreement between mRNA and light output (data not

shown) Of all cell lines SW-480 cells expressing mutant

E and MDA-mb-231 cells expressing mutant F had mRNA

levels that were closest to their respective control cell

lines expressing wild-type luciferases (data not shown)

Although both of these were tested in vivo subsequent

RT-PCR from tumor extracts showed that SW-480 cells

expressing wild-type luciferase consistently lost mRNA

expression when grown in the absence of selection (data

not shown)

Consequently in vivo studies were carried out using

MDA-mb-231 transfected with WT and mutant F lucif-

erases which were shown to have comparable mRNA

levels and representative differences in their steady-state

light output as seen in Figure 3A Transfected WT or

mutant F MDA-mb-231 cells were grown as subcutaneous

xenografts in flanks of SCID mice imaged during tumor

growth All mutant tumors showed greater light intensity

than wild-type tumors SimplePCI software was used

to analyze the light intensity from bioluminescent im-

ages Images were converted to 16-bit TIF images and

were analyzed by implementing a threshold of twice

background to automatically select regions of light out-

put The selected regions were measured for number

of pixels in a region total light intensity over the region

Figure 2 Characterization of mutant luciferases in vitro and in vivo (A) Renaturation of mutant luciferase protein Thermostable mutant luciferase protein was

incubated at 37C for various amounts of time Light was either assayed immediately in the denatured state or allowed to renature at room temperature prior to

being assayed (B) In vitro and in vivo half-lives of wild-type and mutant luciferases In vitro half-lives were determined by IPTG induced bacteria and subsequent

luciferase protein isolation In vivo half-lives were determined from luciferase transfected mammalian tumor cell lines (C) In vivo light output at times following

cycloheximide Typical result showing effects of cyclohexamide inhibiting protein synthesis in cells transfected with thermostable mutant luciferase (D) Effects of

geldanamycin This suggests the role of chaperone activity in the stabilization of luciferase protein Geldanamycin inhibits chaperone activity thereby decreasing light

output without being toxic to the cell



and an average of intensity per pixel was calculated

(Figure 3B) As shown in Figure 3D the light output

from the tumor-expressing mutant F is significantly

higher than that of the tumor-expressing wild-type

luciferase In all mice imaged the mutant tumor pro-

duced greater light intensity than that of the wild-type

tumor on all days imaged Due to slightly varying tumor

growth rates most animals had larger WT tumors than

mutant tumors over the imaging time course whereas

a few had larger mutant F tumors than WT tumors To

verify that tumor size was not the influencing factor MR

images were taken to calculate the tumor volume be-

fore the tumors were palpable This showed that tumor

volume was not the reason for increased light intensity

Figure 3 (A) qRT-PCR Using a primerprobe design luciferase levels were compared to b -actin levels Results of MDA-mb-231 cells transfected with either wild-type or

mutant F luciferase that were used to inoculate mice are shown here Samples are compared at a cycle threshold (30 units) the point at which the slope of the curve

changes At this point the ratio of luciferase to b -actin shows that the expression of luciferase in WT is slightly higher than that of the mutant yet not significantly

different (B) Light output of WT and mutant F from tumors in vivo SimplePCI software was used to analyze images A threshold was set to automatically select regions

of increased intensity The areas selected were calculated for number of pixels in the region and total intensity within the region Area in pixels is an approximation of

tumor volume since tumors were not large enough for caliper measurements until Week 3 (C) In vivo imaging sensitivity Tumor volumes were initially measured by

MR imaging before they were large enough for caliper measurements Tumor volumes here were estimated based on MRI tumor volumes versus number of detectable

pixels for the remainder of the experiment only a percentage of the wild-type tumor is detectable compared to the detectable area of the mutant tumor Due to

increased sensitivity mutant luciferase can be imaged at a much smaller size with much greater intensity Tumor volumes through 4 weeks postinoculation showed

that wild-type tumors were larger than mutant F tumors (D) Bioluminescence of mouse bearing tumors expressing WT and mutant luciferases In all cases the

mutant luciferase showed greater light output than WT In this image MR images were taken to determine tumor volumes showing that although the WT tumor is four

times larger than the mutant tumor the mutant tumor has a higher light intensity greater light per pixel and greater detectable area



The MR tumor volume measurements were also useful

since imaging of tumors was possible more than 2 weeks

before caliper measurements could be made

Discussion

Luciferase is a powerful reporter gene for in vitro

detection of gene expression and has more recently

been used in in vivo bioluminescence imaging Because

the wild-type protein is relatively thermolabile at in vivo

temperatures it was hypothesized that this would limit

the in vivo accumulation of light emitting activity on a

per-cell basis This work has shown that thermostabili-

zation achieved through specific mutations and can lead

to higher steady-state levels of enzyme accumulation

in vivo In all cases heat-induced loss of enzyme activity

was associated with an energy of activation near 340 kJ

mol The relationship between the measured in vivo

half-lives and the apparent activation energy of inacti-

vation is clearly complex For example wild type and

mutant F have essentially the same value of DGDagger

(3619 and 3625 kJmol respectively) yet the in vivo

half-life of mutant F is close to three times that of WT

This may reflect that the irreversible inactivation of

luciferases can occur via several pathways [29ndash32]

making interpretations of apparent DGDagger values difficult

These analyses indicated largely similar activation ener-

gies for enzyme inactivation for both mutants and wild-

type enzymes in the range of 340 kJmol The fact that

the mutant luciferases show greatly increased half-lives

despite little variation in their activation energies for

inactivation is noteworthy This suggests that the mech-

anisms by which the various mutations stabilize the

enzyme do not simply come from enthalpically favorable

interactions increasing the activation energy for in-

activation Although all mutants express longer half-

lives compared to wild-type luciferase both in vitro and

in vivo the in vivo half-lives of all enzymes were signif-

icantly longer than those observed in vitro The mech-

anisms underlying the enhanced in vivo thermostability

are not known One factor is likely the activity of

chaperone proteins which could act to inhibit the

transition from native to molten globule state [33]

Indeed it has previously been shown that chaperones

prolong the half-lives of luciferases both in cell-free and

cell-based assays [27ndash2933] Consistent with this we

have shown that geldanamycin an inhibitor of the

chaperone Hsp90 reduces steady-state luciferase activ-

ities in vivo Notably the in vivo stability can also be

enhanced pharmacologically with phenylbenzothiazole

a competitive inhibitor of luciferin binding [34] Hence

in vivo stability might be coupled to activity yet this is

not explicitly the case as the specific activities of the

thermostable luciferases are comparable to wild type

In all cases thermostabilized luciferases accumulated

to higher levels than wild type as determined by the

relationship between luciferin-induced light output (ac-

tivity) and mRNA levels Notably these data do not

address the effects of these mutations on the reac-

tion kinetics which are being investigated elsewhere

Nonetheless the simplest explanation for the observed

results is the increased accumulation of luciferase en-

zyme with increased half-lives at in vivo temperatures In

either case the empirically observed increase in light

output by the cells and tumors transfected with thermo-

stabilized luciferase increased its efficiency as an in vivo

reporter gene Fewer numbers of cells can be imaged

therefore greatly increasing sensitivity Previously the

detection limit for bioluminescence imaging was deter-

mined to be approximately 1000 cells [126] The in vivo

bioluminescent data here suggest that the minimum

detectable cell number would be closer to 100 cells

Hence the use of thermostabilized luciferases may allow

monitoring of micro-metastases and the early stages of

tumor growth

Acknowledgments

We thank Prof Thomas Baldwin for helpful discussions in the prep-

aration of this manuscript and Ms Merry Warner for help in the pro-

duction of this manuscript The study was supported by the Hughes

and NSF-supported Undergraduate Biology Research Program (RR SM

SM) and NIH CA77375 (BB RJG)

References

[1] Contag CH Jenkins D Contag PR Negrin RS (2000) Use of

reporter genes for optical measurements of neoplastic disease

in vivo Neoplasia 241ndash 52

[2] Contag CH Spilman SD Contag PR Oshiro M Eames B Dennery

P Stevenson DK Benaron DA (1997) Visualizing gene expres-

sion in living mammals using a bioluminescent reporter Photo-

chem Photobiol 66523ndash 531

[3] Contag PR Olomu IN Stevenson DK Contag CH (1998)

Bioluminescent indicators in living mammals Nat Med 4

245ndash 247

[4] Rehemtulla A Stegman LD Cardozo SJ Gupta S Hall DE Contag

CH Ross BD (2000) Rapid and quantitative assessment of cancer

treatment response using in vivo bioluminescence imaging

Neoplasia 2491ndash 495

[5] Wetterwald A van der PG Que I Sijmons B Buijs J Karperien M

Lowik CW Gautschi E Thalmann GN Cecchini MG (2002)

Optical imaging of cancer metastasis to bone marrow A mouse

model of minimal residual disease Am J Pathol 1601143ndash 1153

[6] Shibata T Giaccia AJ Brown JM (2000) Development of a

hypoxia-responsive vector for tumor-specific gene therapy Gene

Ther 7493ndash498

[7] Shibata T Giaccia AJ Brown JM (2002) Hypoxia-inducible



regulation of a prodrug-activating enzyme for tumor-specific

gene therapy Neoplasia 440ndash 48

[8] Shin JH Yi JK Lee YJ Kim AL Park MA Kim SH Lee H Kim

CG (2003) Development of artificial chimerical gene regulatory

elements specific for cancer gene therapy Oncol Rep 10

2063ndash2069

[9] Chinnusamy V Stevenson B Lee BH Zhu JK (2002) Screening

for gene regulation mutants by bioluminescence imaging Sci

STKE 2002L10

[10] Rehemtulla A Hall DE Stegman LD Prasad U Chen G Bhojani

MS Chenevert TL Ross BD (2002) Molecular imaging of gene

expression and efficacy following adenoviral-mediated brain

tumor gene therapy Mol Imaging 143ndash55

[11] Chaudhuri TR Rogers BE Buchsbaum DJ Mountz JM Zinn KR

(2001) A noninvasive reporter system to image adenoviral-

mediated gene transfer to ovarian cancer xenografts Gynecol

Oncol 83432ndash 438

[12] Brun S Faucon-Biguet N Mallet J (2003) Optimization of

transgene expression at the posttranscriptional level in neural

cells Implications for gene therapy Mol Ther 7782ndash789

[13] Siemens DR Crist S Austin JC Tartaglia J Ratliff TL (2003)

Comparison of viral vectors Gene transfer efficiency and tissue

specificity in a bladder cancer model J Urol 170979ndash 984

[14] Nunn C Feuerbach D Lin X Peter R Hoyer D (2002) Pharma-

cological characterisation of the goldfish somatostatin sst5 re-

ceptor Eur J Pharmacol 436173ndash 186

[15] Kimm-Brinson KL Moeller PD Barbier M Glasgow H Jr

Burkholder JM Ramsdell JS (2001) Identification of a P2X7

receptor in GH(4)C(1) rat pituitary cells A potential target for a

bioactive substance produced by Pfiesteria piscicida Environ

Health Perspect 109457ndash 462

[16] Catania JM Parrish AR Kirkpatrick DS Chitkara M Bowden GT

Henderson CJ Wolf CR Clark AJ Brendel K Fisher RL Gandolfi

AJ (2003) Precision-cut tissue slices from transgenic mice as an

in vitro toxicology system Toxicol In Vitro 17201ndash 205

[17] Billard P DuBow MS (1998) Bioluminescence-based assays for

detection and characterization of bacteria and chemicals in

clinical laboratories Clin Biochem 311ndash 14

[18] Bitton G Koopman B (1992) Bacterial and enzymatic bioassays

for toxicity testing in the environment Rev Environ Contam

Toxicol 1251 ndash22

[19] Greer LF Szalay AA III (2002) Imaging of light emission from the

expression of luciferases in living cells and organisms A review

Luminescence 1743ndash 74

[20] Hastings JW (1996) Chemistries and colors of bioluminescent

reactions A review Gene 1735 ndash11

[21] Viviani VR (2002) The origin diversity and structure func-

tion relationships of insect luciferases Cell Mol Life Sci 59

1833ndash 1850

[22] Lall AB Ventura DS Bechara EJ de Souza JM Colepicolo-Neto P

Viviani VR (2000) Spectral correspondence between visual

spectral sensitivity and bioluminescence emission spectra in the

click beetle Pyrophorus punctatissimus (Coleoptera Elateridae)

J Insect Physiol 461137ndash 1141

[23] Viviani VR Silva AC Perez GL Santelli RV Bechara EJ Reinach FC

(1999) Cloning and molecular characterization of the cDNA for

the Brazilian larval click-beetle Pyrearinus termitilluminans

luciferase Photochem Photobiol 70254ndash 260

[24] Schmitter RE Njus D Sulzman FM Gooch VD Hastings JW

(1976) Dinoflagellate bioluminescence A comparative study of

in vitro components J Cell Physiol 87123ndash 134

[25] Day RN Kawecki M Berry D (1998) Dual-function reporter

protein for analysis of gene expression in living cells BioTech-

niques 25848ndash4 856

[26] Leclerc GM Boockfor FR Faught WJ Frawley LS (2000) De-

velopment of a destabilized firefly luciferase enzyme for mea-

surement of gene expression BioTechniques 29590ndash596 598

[27] Souren JE Wiegant FA van Hof P van Aken JM van Wijk R

(1999) The effect of temperature and protein synthesis on the

renaturation of firefly luciferase in intact H9c2 cells Cell Mol Life

Sci 551473ndash1481

[28] Souren JE Wiegant FA van Wijk R (1999) The role of hsp70

in protection and repair of luciferase activity in vivo experi-

mental data and mathematical modelling Cell Mol Life Sci 55

799ndash 811

[29] Herbst R Schafer U Seckler R (1997) Equilibrium intermediates

in the reversible unfolding of firefly (Photinus pyralis) luciferase

J Biol Chem 2727099ndash7105

[30] Herbst R Gast K Seckler R (1998) Folding of firefly (Photinus

pyralis) luciferase Aggregation and reactivation of unfolding

intermediates Biochemistry 376586ndash 6597

[31] Baldwin TO Chen LH Chlumsky LJ Devine JH Ziegler MM

(1989) Site-directed mutagenesis of bacterial luciferase Analysis

of the lsquolsquoessentialrsquorsquo thiol J Biolumin Chemilumin 440ndash48

[32] Chen LH Baldwin TO (1989) Random and site-directed muta-

genesis of bacterial luciferase Investigation of the aldehyde

binding site Biochemistry 282684ndash 2689

[33] Flynn GC Beckers CJ Baase WA Dahlquist FW (1993)

Individual subunits of bacterial luciferase are molten globules

and interact with molecular chaperones Proc Natl Acad Sci

USA 9010826ndash 10830

[34] Thompson JF Hayes LS Lloyd DB (1991) Modulation of firefly

luciferase stability and impact on studies of gene regulation

Gene 103171ndash 177



(HBSS) 54 mM KCl 04 mM KH2PO4 42 mM NaHCO3

137 mM NaCl 03 mM Na2HPO4 In order to gener-

ate HBSS at different pH values pH was adjusted by

adding either HCl or NaOH to the desired pH Native

firefly luciferase-recombinant protein was purchased

from RampD Systems (Minneapolis MN) (700-LF-01M)

LuciferinmdashD-luciferin potassium salt was a generous gift

from Brian Ross (University of Ann Arbor Michigan)

Luciferase Assay reagent (LAR) and Cell Culture Lysis Re-

agent (CCLR) were purchased from Promega (Madison

WI) Luciferase mutants were cloned into a pET23a(+)

bacterial expression vector at the NdeI and SalI sites of

the MCS and maintained in Escherichia coli B121 DE3

Wild-type luciferase was isolated from pGEM-luc from

Promega The mammalian expression vector used was

pcDNA31() from Invitrogen (Carlsbad CA) with a se-

lectable neomycin resistance gene

Production of Mutant Luciferases from Bacteria

Preliminary experiments were done to optimize for

induction time DH5a bacteria were transformed with a

pET23a(+)mutant luciferase construct Colonies of

bacteria were isolated then grown in liquid cultures of

LB broth (Sigma L 3152) in an orbital shaking incubator

One milliliter of cell suspension was removed the bacte-

rial cells pelleted then resuspended in 1 mL HBSS

pH 78 This sample was placed in the luminometer a

background reading was taken D-luciferin was added

and the light intensity was recorded IPTG was then

added to the remainder of the culture to a final concen-

tration of 01 mM and cultures were left in an orbital

shaking incubator Samples were then removed at multi-

ple time points post-IPTG induction and the assay pro-

cess was repeated Peak induction was seen between

1 and 2 hr In vitro experiments were performed by

isolating the mutant luciferase protein from bacterial

cultures In an orbital shaking incubator 2-mL starter

cultures were grown from isolated colonies incubated

overnight and then transferred to 100 mL LB broth After

incubating for 2 hr cells were induced with IPTG Two

hours post-induction cells were harvested and washed a

lysozyme buffer (9 mgmL lysozyme in PBS) was added

and the cells were subjected to a freezendashthaw cycle after

addition of Triton X-100 Samples were spun and super-

natant aliquoted then frozen for use with in vitro tem-

perature half-life assays

Mammalian Cell Culture

All mammalian cell lines were grown in DMEF-12

media (Sigma D0547) with 10 FBS Cells were pas-

saged weekly The parental cell lines MDA-mb-231

(metastatic breast) and SW-480 (colon) were plated into

six-well plates or 10-cm dishes for transfections Stable

cell lines were created by adding selection media (G418

sulfate Geneticin 400 mgmL Invitrogen) and cells

maintained were under selection Cells for mouse inoc-

ulations were expanded equally into several T-75 flasks

one flask was trypsinized and counted in an appropriate

number of flasks cells were scraped centrifuged and

resuspended in a volume of 11 salinecell suspension to

Matrigel (BD Biosciences Franklin Lakes NJ) so that a

100-mL inoculum contains 3 106 cells

Preparation of Mammalian Expression Vectors

DNA was isolated from colonies of B121 DE3 E coli

transformed with a bacterial expression vector carrying

one of six different mutant forms of luciferase that were

previously generated by a random mutagenesis screen in

yeast These mutant sequences were cloned into the

NdeI and SalI sites of the MCS of pET23a(+) (Tisi

unpublished) Competent DH5a cells were then trans-

formed with the various mutants designated A-F (see

Table 1) Each mutant DNA was isolated digested and

ligated into a mammalian expression vector All mutants

were transferred into the pcDNA31() mammalian

expression vector at ApaI and XhoI The wild-type

luciferase was transferred from pGEM-luc (Promega) to

pcDNA31() also at ApaI and XhoI SW-480 colon can-

cer cells were transiently transfected using FuGene-6

Table 1 Thermostability of Luciferases

Luciferase Mutations In vitro In vivo

T05 at 37C DG+ (kJmol) T05 at 37C

WT NA 306 36190 493 plusmn 214

Mutant A E345K T214A 85 plusmn 10 30973 1363 plusmn 297

Mutant B E345K I232A

T214A

155 plusmn 145 35340 1419 plusmn 157

Mutant C E345K A215L 736 plusmn 182 40365 1604 plusmn 353

Mutant D E345K I232A

T214A F295L

S420T

724 plusmn 31 27074 1281 plusmn 114

Mutant E E345K A215L

I232A T214A

F295L

821 plusmn 85 28068 2494 plusmn 587

Mutant F E345K A215L

I232A T214A

751 plusmn 981 36246 1355 plusmn 169

For in vitro studies luciferase mutants were cloned expressed and purified

from DH5a bacteria and incubated at various temperatures (from 21C to 45C)

for various times prior to assay at 23C Plots of enzyme activity (light output)

versus time were fitted to first-order exponential to obtain half-lives Arrhenius

plot of half-lives versus temperature was generated for each mutant to

determine the DG+ associated with the inactivation process For in vivo

studies SW-480 cells stably expressing each of these luciferases were treated

with 001 mM cycloheximide at 37C and extracted at time points thereafter

The resulting plot of light output versus time was fitted to a first-order

exponential to yield half-life



















































qRT-PCR
























Results


























































































for wild type










































In Vivo Imaging





























not shown)
























































Discussion





































































tumor growth

Acknowledgments






References










245ndash 247











Ther 7493ndash498









2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830






















































qRT-PCR
























Results


























































































for wild type










































In Vivo Imaging





























not shown)
























































Discussion





































































tumor growth

Acknowledgments






References










245ndash 247











Ther 7493ndash498









2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830




































































for wild type










































In Vivo Imaging





























not shown)
























































Discussion





































































tumor growth

Acknowledgments






References










245ndash 247











Ther 7493ndash498









2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830
























not shown)
























































Discussion





































































tumor growth

Acknowledgments






References










245ndash 247











Ther 7493ndash498









2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830





































Discussion





































































tumor growth

Acknowledgments






References










245ndash 247











Ther 7493ndash498









2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830











2063ndash2069



STKE 2002L10








































1833ndash 1850






















Sci 551473ndash1481




799ndash 811
















USA 9010826ndash 10830






Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence

Documents

Transcript of Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence